Methods for producing a complex transgenic trait locus

ABSTRACT

Methods for producing in a plant a complex transgenic trait locus comprising at least two altered target sequences in a genomic region of interest are disclosed. The methods involve the use of two or more double-strand-break-inducing agents, each of which can cause a double-strand break in a target sequence in the genomic region of interest which results in an alteration in the target sequence. Also disclosed are complex transgenic trait loci in plants. A complex transgenic trait locus comprises at least two altered target sequences that are genetically linked to a polynucleotide of interest. Plants, plant cells, plant parts, and seeds comprising one or more complex transgenic trait loci are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/007,529 filed on 13 Jun. 1018, which is a continuation of U.S. patentapplication Ser. No. 13/427,138, now granted as U.S. Pat. No. 10,030,245granted on 24 Jul. 2018, which itself claims the benefit of U.S.Provisional Patent Application No. 61/499,443, filed Jun. 11, 2011 andU.S. Provisional Patent Application No. 61/466,602, filed Mar. 23, 2011;all of which are hereby incorporated herein in their entirety byreference.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing is submitted as a text file namedBB1990-US-CNT[2]_SeqListing_ST25.txt, created on 8 Jul. 2020, having asize of 154,570 bytes, and is hereby incorporated by reference pursuantto 37 C.F.R. § 1.52(e)(5).

FIELD OF INVENTION

The invention relates to the field of plant molecular biology, inparticular, to methods for altering the genome of a plant cell.

BACKGROUND

Recombinant DNA technology has made it possible to insert foreign DNAsequences into the genome of an organism, thus, altering the organism'sphenotype. The most commonly used plant transformation methods areAgrobacterium infection and biolistic particle bombardment in whichtransgenes integrate into a plant genome in a random fashion and in anunpredictable copy number. Thus, efforts are undertaken to controltransgene integration in plants.

One method for inserting or modifying a DNA sequence involves homologousDNA recombination by introducing a transgenic DNA sequence flanked bysequences homologous to the genomic target. U.S. Pat. No. 5,527,695describes transforming eukaryotic cells with DNA sequences that aretargeted to a predetermined sequence of the eukaryote's DNA.Specifically, the use of site-specific recombination is discussed.Transformed cells are identified through use of a selectable markerincluded as a part of the introduced DNA sequences.

It was shown that artificially induced site-specific genomicdouble-stranded breaks in plant cells were repaired by homologousrecombination with exogenously supplied DNA using two differentpathways. (Puchta et al., (1996) Proc. Natl. Acad. Sci. USA93:5055-5060; U.S. Patent Application Publication No. 2005/0172365A1published Aug. 4, 2005; U.S. Patent Application Publication No.2006/0282914 published Dec. 14, 2006; WO 2005/028942 published Jun. 2,2005).

Since the isolation, cloning, transfer and recombination of DNAsegments, including coding sequences and non-coding sequences, is mostconveniently carried out using restriction endonuclease enzymes. Muchresearch has focused on studying and designing endonucleases such as WO2004/067736 published Aug. 12, 2004; U.S. Pat. No. 5,792,632 issued toDujon et al., Aug. 11, 1998; U.S. Pat. No. 6,610,545 B2 issued to Dujonet al., Aug. 26, 2003; Chevalier et al., (2002) Mol Cell 10:895-905;Chevalier et al., (2001) Nucleic Acids Res 29:3757-3774; Seligman etal., (2002) Nucleic Acids Res 30:3870-3879.

Although a plethora of approaches have been developed to target aspecific site for modification in the genome of a plant, there stillremains a need for methods for producing a fertile plant, having analtered genome comprising two or more site-specific modifications indefined region of the genome of the plant.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for producing in a plant acomplex transgenic trait locus comprising at least two altered targetsequences in a genomic region of interest. The methods involve selectinga genomic region in a plant that comprises a first target sequence and asecond target sequence and then providing a firstdouble-strand-break-inducing agent and a seconddouble-strand-break-inducing agent. The firstdouble-strand-break-inducing agent is capable of inducing a firstdouble-strand break in DNA comprising the first target sequence, and thesecond double-strand-break-inducing agent is capable of inducing asecond double-strand break in DNA comprising the second target sequence.The methods further involve contacting at least one plant cell with thefirst double-strand-break-inducing agent, identifying a cell comprisinga first alteration at the first target sequence, and then recovering afirst fertile plant from the cell comprising the first alteration. Thefirst fertile plant also comprises the first alteration. Additionally,the methods involve contacting at least one plant cell with the seconddouble-strand-break-inducing agent, identifying a cell comprising asecond alteration at the second target sequence, and then recovering asecond fertile plant from the cell comprising the second alteration. Themethods further involve obtaining a fertile progeny plant from thesecond fertile plant, wherein the fertile progeny plant comprises boththe first and second alterations in physical linkage.

In a first embodiment of the methods for producing in a plant a complextransgenic trait locus, the fertile progeny plant is obtained bycrossing the first fertile plant and the second fertile plant andselecting the fertile progeny plant comprising both the first and secondalterations in physical linkage.

In second embodiment, a cell of the first fertile plant, or progenythereof comprising the first alteration, is contacted with the seconddouble-strand-break-inducing agent.

In third embodiment, the complex transgenic trait locus furthercomprises at least one polynucleotide of interest in the genomic regionof interest. Such a polynucleotide of interest can be, for example, atransgene, a native gene, and a gene that was a native gene prior to atargeted mutation therein.

In a fourth embodiment, the first alteration comprises insertion of afirst DNA sequence of interest, or part thereof, into the first targetsequence, and/or the second alteration comprises insertion of a secondDNA sequence of interest, or part thereof, into the second targetsequence. Such a first and/or a second DNA sequence of interest can be,for example, a DNA for gene silencing, a DNA encoding a phenotypicmarker and a DNA encoding a protein providing an agronomic advantage.

In a fifth embodiment, the first and second double-strand-break-inducingagents are selected from the group consisting of an endonuclease, a zincfinger nuclease, or a TAL effector nuclease.

In a sixth embodiment, the endonuclease is modified to specifically cutat the first target sequence or at the second target sequence and nolonger cuts at its wild-type endonuclease target sequence.

In a seventh embodiment, the first target sequence and the second targetsequence are separated from each other by about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8., 0.9, 1, 2, 3, 4, or 5 centimorgans (cM) in the genome ofthe plant.

In an eighth embodiment, the methods can involve crossing the fertileprogeny plant with an additional fertile plant that comprises at least athird altered target sequence in the genomic region of interest and thenselecting from the crossing a fertile progeny plant comprising the firstalteration, the second and the at least third alteration in physicallinkage. Like the first and second altered target sequences, the thirdaltered target sequence originated from a third target sequence that isrecognized and cleaved by a third double-strand-break-inducing agent.

Additionally provided are complex trait loci in plants produced by themethods of the invention and plants, plant cells, plant parts, and seedsthereof comprising at least one complex transgenic trait locus of theinvention.

The present invention further provides a complex transgenic trait locuscomprising at least two altered target sequences that are geneticallylinked in the genome of a plant to a polynucleotide of interest. Suchaltered target sequences originated from a corresponding target sequencethat is recognized and cleaved by a double-strand-break-inducing agent.The altered target sequences comprise an alteration such as, forexample, replacement of at least one nucleotide in the target sequence,a deletion of at least one nucleotide in the target sequence, aninsertion of at least one nucleotide in the target sequence, or anycombination thereof. The polynucleotide interest can be, for example, atransgene, a native gene, and a mutated gene. The present inventionfurther provides plants, plant parts, plant cells, and seeds comprisingat least one complex transgenic trait locus of the invention.

In an embodiment of the complex transgenic trait locus of the invention,at least one altered target sequence comprises a recombinant DNAmolecule. Recombinant DNA molecules include, but are not limited to, aDNA for gene silencing, a DNA encoding a phenotypic marker, and a DNAencoding a protein providing an agronomic advantage.

In another embodiment, the two altered target sequences of the complextransgenic trait locus are located within about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8., 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11 or up to 21centimorgan (cM) of the polynucleotide of interest.

The invention provides plants, plant parts, plant cells, and seedscomprising at least one complex transgenic trait locus of the invention.

Additionally provided is an alternative method for producing in a planta complex transgenic trait locus comprising at least two altered targetsequences in a genomic region of interest. This method involvesobtaining a first fertile plant comprising a first altered targetsequence at the genomic region of interest and a second fertile plantcomprising a second altered target sequence at the genomic region ofinterest. In this method, the first altered target sequence originatedfrom a first target sequence that is recognized and cleaved by a firstdouble-strand-break-inducing agent, and the second altered targetsequence originated from a second target sequence that is recognized andcleaved by a second double-strand-break-inducing agent. The alternativemethod further involves crossing the first fertile plant and the secondfertile plant, and then selecting from the crossing a fertile progenyplant comprising the first alteration and the second alteration inphysical linkage.

Also provided are plants produced by the second method of the inventionand plant cells, plant parts, and seeds thereof comprising at least onecomplex transgenic trait locus.

In another embodiment, the present invention provides a plant comprisingan expression construct, which comprises a promoter operably linked to anucleotide sequence encoding an endonuclease. The endonuclease iscapable of specifically binding to and creating a double strand break ina target sequence selected from the group consisting of SEQ ID NO:1, 2,3, 4, 5, 6, 7, 8, 68, 69, 70, 71, 72, 73, 74, 75, 76, and 77, whereinthe promoter is capable of driving expression of an operably linkednucleotide sequence in a plant cell. The nucleotide sequence encodingthe endonuclease can comprise a coding sequence of a DNA binding domainof an endonuclease, wherein the coding sequence comprises nucleotides100-261 and nucleotides 850-1011 of SEQ ID NO:9, 10, 11, 12, 13, 14, 15,16, 78, 79, 80, 81, 82 or 83; ora degenerate coding sequence thereof.Preferably, the nucleotide sequence encoding the endonuclease is anucleotide sequence selected from the group consisting of SEQ ID NO:9,10, 11, 12, 13, 14, 15, 16, 78, 79, 80, 81, 82, and 83.

In yet another embodiment of the invention, a plant of the inventioncomprises at least one altered target sequence, wherein the at least onealtered target sequence originated from a corresponding target sequencethat was recognized and cleaved by a double-strand break-inducing agent.In this embodiment, the altered target sequence is in a genomic regionof interest that extends from: the target sequence set forth in SEQ IDNO: 4 to the target sequence set forth in SEQ ID NO: 2; the targetsequence set forth in SEQ ID NO: 5 to the target sequence set forth inSEQ ID NO: 8; or the target sequence set forth in SEQ ID NO: 68 to thetarget sequence set forth in SEQ ID NO: 77. Such a plant of theinvention can be produced by a method comprising providing at least onedouble-strand-break-inducing agent that is capable of inducing adouble-strand break in DNA comprising a target sequence, wherein thetarget sequence is in a genomic region of interest that extends from:the target sequence set forth in SEQ ID NO: 4 to the target sequence setforth in SEQ ID NO: 2; the target sequence set forth in SEQ ID NO: 5 tothe target sequence set forth in SEQ ID NO: 8; or the target sequenceset forth in SEQ ID NO: 68 to the target sequence set forth in SEQ IDNO: 77. The method further comprises contacting at least one plant cellwith the double-strand-break-inducing agent, identifying a cellcomprising an alteration at the target sequence, and recovering afertile plant comprising the alteration. In one embodiment of thismethod, the double-strand-break-inducing agent is encoded by anucleotide sequence comprising a coding sequence of a DNA binding domainof an endonuclease, and wherein the coding sequence is selected from thegroup consisting of nucleotides 100-261 and nucleotides 850-1011 of SEQID NO: 9, 10, 11, 12, 13, 14, 15, 16, and 80, and degenerate codingsequences thereof. In another embodiment of this method, thedouble-strand-break-inducing agent is encoded by a nucleotide sequencecomprising a coding sequence of a DNA binding domain of an endonuclease,and wherein the coding sequence is selected from the group consisting ofnucleotides 100-261 and nucleotides 661-822 of SEQ ID NO: 78, 79, 81, 82and 83, and degenerate coding sequences thereof. In another embodimentof this method, the double-strand-break-inducing agent is encoded by anucleotide sequence is selected from the group consisting of SEQ ID NO:9, 10, 11, 12, 13, 14, 15, 16, 78, 79, 80, 81, 82, and 83.

Additional embodiments of the methods and compositions of the presentinvention are disclosed below.

BRIEF DESCRIPTION OF THE DRAWINGS AND THE SEQUENCE LISTING

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing, whichform a part of this application. The sequence descriptions and sequencelisting attached hereto comply with the rules governing nucleotide andamino acid sequence disclosures in patent applications as set forth in37 C.F.R. §§ 1.821-1.825. The sequence descriptions contain the threeletter codes for amino acids as defined in 37 C.F.R. §§ 1.821-1.825,which are incorporated herein by reference.

FIGURES

FIG. 1. DNA double-strand break induced DNA alteration of an endogenoustarget site. FIG. 1A A generalized endogenous target site with flankinggenomic DNA sequences designated as DNA 1 and DNA 2 which can be used asDNA exchange regions by homologous recombination. FIG. 1B A generalizedDNA construct that can be used to express a DNA endonuclease torecognize and cleave the endogenous target site. The DNA endonucleasegene can be physically linked to the donor DNA described in FIG. 1C orFIG. 1D, or substituted by other double-strand break inducing agents.FIG. 1C A generalized donor DNA construct having two regions DNA1 andDNA 2 of homology to the genomic target which flank a polynucleotide ofinterest and/or marker gene. FIG. 1D A generalized donor DNA constructthat does not have regions of homology to the genomic target to flank apolynucleotide of interest and/or marker gene. Insertion of the DNAfragment will produce an insertion of the polynucleotide of interest ator near the recognition site. FIG. 1E One expected outcome when thepolynucleotide of interest and/or marker gene of donor constructdescribed in FIG. 1C or FIG. 1D is inserted at the endogenous targetsite by homologous recombination or non-homologous recombination,respectively. FIG. 1F Another outcome when the endogenous target site isaltered by a deletion during the repair of the DNA double-strand breakcleaved by the DNA endonuclease. The polynucleotide of interest and/ormarker gene of donor construct described in FIG. 1C or FIG. 1D can beinserted at unrelated sites by random DNA integration. FIG. 1G Anotheroutcome when the endogenous target site is altered by the insertion ofan unrelated DNA during the repair of the DNA double-strand breakscleaved by the DNA endonuclease. The polynucleotide of interest and/ormarker gene of donor construct described in FIG. 1C or FIG. 1D can beinserted at unrelated sites by random DNA integration.

FIG. 2. Genetic distance between target sites and transgene of interest.

FIG. 3. FIG. 3A: Schematic diagram of PCR assays to detect TS21 targetsite modifications and transgenic integrations. FIG. 3B: Alignment ofaltered target sequences of selected TS21 transgenic event.

FIG. 4. FIG. 4A: Alignment of altered target sequences of selected TS5transgenic events. FIG. 4B: Alignment of altered target sequences ofselected TS14 transgenic events

FIG. 5. Gene integration by homologous recombination enabled bydouble-strand breaks with custom designed meganuclease.

FIG. 6. Location of target sites near a herbicide resistant transgenicevent in soybean.

FIG. 7. Use of cluster of meganuclease target sites for stacking ofmultiple traits either by sequential transformation or genetic crosses.

FIG. 8. The locations of various MHP target sites surrounding atransgenic DNA of interest integration site in a maize plant. Solidblack rectangles represent BAC clones. Names and numbers in each box arethe target sites. Arrows from box to BAC indicated the target siteaffiliated to BAC clones. Numbers and arrows on the bottom of the figureindicate the genetic distance of the target sites relative to theinsertion location of the transgenic DNA of interest. As indicated atthe top of the figure, the physical distance is about 1.8 Mb nucleotidesin this region of the maize chromosome.

FIG. 9. FIG. 9A: Outline of PCR screening for integration of donor atMHP14 target site (donor was PHP44779) FIG. 9B: PCR of MHP14 events:B1-B12 junction PCR with primers 146773/146775; b1-b12 junction PCR withprimers 146772/146778. Two events (B2 and B5) were positive for bothjunctions PCR. The arrows indicate the locations corresponding to thevarious primers used.

FIG. 10. FIG. 10A: Schematic outline of PCR to confirm ubi:mopat:pinllcassette integration at the endogenous MHP14 target . FIG. 10B: Long PCRon TO plants from three events showed integration at the target site.The plant A5 was from event #1, A6-A8 event #2, and C4-C6 event #3. CKP:positive control from callus DNA. FIG. 10B: The left panel shows theresults of junction PCR on the HR1 side using a genomic primer (146775)and a moPAT primer (mopatR2). The right panel shows the results ofjunction PCR on the HR2 side with a moPAT primer (mopatF2) a genomicprimer (146772). The arrows on FIG. 10A indicate the locationscorresponding to the various primers used.

FIGS. 11A-11D. Alignment of fragments from the plant-optimizednucleotide sequences of meganucleases comprising the nucleotides 100-261and nucleotides 850-1011 of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16,and 80, and the nucleotides 100-261 and nucleotides 661-822 of SEQ IDNO: 78, 79, 81, 82 and 83 . FIG. 11A shows SEQ ID NOs: 9-16 and 78-80sequence positions 60-180, FIG. 11B shows SEQ ID NOs: 9-16 and 78-80sequence positions 180-300, FIG. 11C shows SEQ ID NOs: 9-16 and 78-80sequence positions 840-960, and FIG. 11D shows SEQ ID NOs: 9-16 and78-80 sequence positions 960-1020.

SEQUENCES

SEQ ID NO: 1 is the nucleotide sequence of the TS21 target site insoybean genome.

SEQ ID NO: 2 is the nucleotide sequence of the TS14 target site insoybean genome.

SEQ ID NO: 3 is the nucleotide sequence of the TS30 target site insoybean genome.

SEQ ID NO: 4 is the nucleotide sequence of the TS5 target site insoybean genome.

SEQ ID NO: 5 is the nucleotide sequence of the TS7 target site insoybean genome.

SEQ ID NO: 6 is the nucleotide sequence of the TS4 target site insoybean genome.

SEQ ID NO: 7 is the nucleotide sequence of the TS22 target site insoybean genome.

SEQ ID NO: 8 is the nucleotide sequence of the TS24 target site insoybean genome.

SEQ ID NO: 9 is the plant-optimized nucleotide sequence of the TS21meganuclease containing a nuclear target site and an ST-LS1 intron.

SEQ ID NO: 10 is the plant-optimized nucleotide sequence of the TS14meganuclease containing a nuclear target site and an ST-LS1 intron.

SEQ ID NO: 11 is the plant-optimized nucleotide sequence of the TS30meganuclease containing a nuclear target site and an ST-LS1 intron.

SEQ ID NO: 12 is the plant-optimized nucleotide sequence of the TS5meganuclease containing a nuclear target site and an ST-LS1 intron.

SEQ ID NO: 13 is the plant-optimized nucleotide sequence of the TS7meganuclease containing a nuclear target site and an ST-LS1 intron.

SEQ ID NO: 14 is the plant-optimized nucleotide sequence of the TS4meganuclease containing a nuclear target site and an ST-LS1 intron.

SEQ ID NO: 15 is the plant-optimized nucleotide sequence of the TS22meganuclease containing a nuclear target site and an ST-LS1 intron.

SEQ ID NO: 16 is the plant-optimized nucleotide sequence of the TS24meganuclease containing a nuclear target site and an ST-LS1 intron.

SEQ ID NO: 17 is the homologous region 1 (HR1) of the TS21 target site.

SEQ ID NO: 18 is the homologous region 2 (HR2) of the TS21 target site.

SEQ ID NO: 19 is the HR1 of the TS14 target site.

SEQ ID NO: 20 is the homologous region 2 of the TS14 target site.

SEQ ID NO: 21 is the HR1 of the TS30 target site.

SEQ ID NO: 22 is the homologous region 2 of the TS30 target site.

SEQ ID NO: 23 is the HR1 of the TS5 target site.

SEQ ID NO: 24 is the homologous region 2 of the TS5 target site.

SEQ ID NO: 25 is the HR1 of the TS7 target site.

SEQ ID NO: 26 is the homologous region 2 of the TS7 target site.

SEQ ID NO: 27 is the HR1 of the TS4 target site.

SEQ ID NO: 28 is the homologous region 2 of the TS4 target site.

SEQ ID NO: 29 is the HR1 of the TS22 target site.

SEQ ID NO: 30 is the homologous region 2 of the TS22 target site.

SEQ ID NO: 31 is the HR1 of the TS24 target site.

SEQ ID NO: 32 is the homologous region 2 of the TS24 target site.

SEQ ID NO: 33 is the plant-optimized nucleotide sequence of the TS21meganuclease without a ST-LS1 intron.

SEQ ID NO: 34 is the amino acid sequence of the SV40 nuclearlocalization signal.

SEQ ID NO: 35: is the nucleotide sequences of expression cassetteRTW317, comprising the TS21 meganuclease plant optimized sequencewithout an intron and operably linked to the soybean EF1A promoter.

SEQ ID NO: 36 is the nucleotide sequences of expression cassette RTW322,comprising the TS21 meganuclease plant optimized sequence with an intronand operably linked to the soybean EF1A promoter.

SEQ ID NO: 37 is the nucleotide sequence of RTW328A, which is the repairDNA fragment for TS21 meganuclease.

SEQ ID NO:38 is the nucleotide sequence of TS21 qPCR forward primerMega21-190F.

SEQ ID NO:39 is the nucleotide sequence of TS21 qPCR reverse primerMega21-301R.

SEQ ID NO:40 is the nucleotide sequence of TS21 qPCR probe mega21-250T.The fluorescent probe is labeled with FAM quenched with MGB.

SEQ ID NO:41 is the nucleotide sequence of TS14 qPCR forward primerMega14-13F.

SEQ ID NO:42 is the nucleotide sequence of TS14 qPCR reverse primerMega14-128R.

SEQ ID NO:43 is the nucleotide sequence of TS14 qPCR probe Mega14-85T.The fluorescent probe is labeled with FAM quenched with MGB.

SEQ ID NO:44 is the nucleotide sequence of TS30 qPCR forward primerMega30-30F.

SEQ ID NO:45 is the nucleotide sequence of TS30 qPCR reverse primerMega30-87R.

SEQ ID NO:46 is the nucleotide sequence of TS30 qPCR probe Mega30-52T.The fluorescent probe is labeled with FAM quenched with MGB.

SEQ ID NO:47 is the nucleotide sequence of TS5 qPCR forward primerMega5-F1.

SEQ ID NO:48 is the nucleotide sequence of TS5 qPCR reverse primerMega5-R1.

SEQ ID NO:49 is the nucleotide sequence of TS5 qPCR probe Mega5-T1. Thefluorescent probe is labeled with FAM quenched with MGB.

SEQ ID NO:50 is the nucleotide sequence of the sense primer, WOL133,which is upstream of the TS21 target site in the soybean genome.

SEQ ID NO:51 is the nucleotide sequence of the antisense primer, WOL134,which is downstream of the TS21 target site in the soybean genome.

SEQ ID NO:52 is the nucleotide sequence of the sense primer, WOL190which is further upstream of the TS21 target site beyond the TS21 HR1fragment in the soybean genome.

SEQ ID NO:53 is the nucleotide sequence of the antisense primer, WOL242,which is specific to the hygromycin coding sequences.

SEQ ID NO:54 is the nucleotide sequence of the sense primer, WOL153,which is specific to the NOS Terminator.

SEQ ID NO:55 is the nucleotide sequence of the antisense primer, WOL247,which is further downstream of the TS21 target site beyond the TS21 HR2fragment in the soybean genome.

SEQ ID NO:56 is the nucleotide sequence of the sense primer, WOL121,which is upstream of the TS14 target site in the soybean genome.

SEQ ID NO:57 is the nucleotide sequence of the antisense primer, WOL150,which is downstream of the TS21 target site in the soybean genome.

SEQ ID NO:58 is the nucleotide sequence of the sense primer, WOL192,which is further upstream of the TS14 target site beyond the TS14 HR1fragment in the soybean genome.

SEQ ID NO:59 is the nucleotide sequence of the antisense primer, WOL193,which is further downstream of the TS14 target site beyond the TS14 HR2fragment in the soybean genome.

SEQ ID NO:60 is the nucleotide sequence of the sense primer, WOL113,which is upstream of the TS30 target site in the soybean genome.

SEQ ID NO:61 is the nucleotide sequence of the antisense primer, WOL114,which is downstream of the TS30 target site in the soybean genome.

SEQ ID NO:62 is the nucleotide sequence of the sense primer, WOL194,which is further upstream of the TS30 target site beyond the TS30 HR1fragment in the soybean genome.

SEQ ID NO:63 is the nucleotide sequence of the antisense primer, WOL195,which is further downstream of the TS30 target site beyond the TS30 HR2fragment in the soybean genome.

SEQ ID NO:64 is the nucleotide sequence of the sense primer, WOL105,which is upstream of the TS5 target site in the soybean genome.

SEQ ID NO:65 is the nucleotide sequence of the antisense primer, WOL144,which is downstream of the TS5 target site in thesoybean genome.

SEQ ID NO:66 is the nucleotide sequence of the sense primer, WOL196,which is further upstream of the TS5 target site beyond the TS5 HR1fragment in the soybean genome.

SEQ ID NO:67 is the nucleotide sequence of the antisense primer, WOL197,which is further downstream of the TS5 target site beyond the TS5 HR2fragment in the soybean genome.

SEQ ID NO:68 is the nucleotide sequence of the MHP1 target site in themaize genome.

SEQ ID NO:69 is the nucleotide sequence of the MHP14 target sitesequence in the maize genome.

SEQ ID NO:70 is the nucleotide sequence of the MHP32 target sitesequence in the maize genome.

SEQ ID NO:71 is the nucleotide sequence of the MHP42 target sitesequence in the maize genome.

SEQ ID NO:72 is the nucleotide sequence of the MHP55 target sitesequence in the maize genome.

SEQ ID NO:73 is the nucleotide sequence of the MHP67 target sitesequence in the maize genome.

SEQ ID NO:74 is the nucleotide sequence of the MHP77 target sitesequence in the maize genome.

SEQ ID NO:75 is the nucleotide sequence of the MHP98 target sit sequencein the maize genome.

SEQ ID NO:76 is the nucleotide sequence of the MHP107 target sitesequence in the maize genome.

SEQ ID NO: 77 is the nucleotide sequence of the MHP115 target sitesequence in the maize genome.

SEQ ID NO:78 is the plant-optimized nucleotide sequence of MHP14comprising a nuclear localization signal and lacking an intron.

SEQ ID NO:79is the plant-optimized nucleotide sequence of the MHP14+comprising a nuclear localization signal and lackingan intron.

SEQ ID NO:80 is the plant-optimized nucleotide sequence of MHP55comprising a nuclear localization signal and an intron.

SEQ ID NO:81 is the plant-optimized nucleotide sequence of MHP55comprising a nuclear localization signal and lackingan intron.

SEQ ID NO:82 is the plant-optimized nucleotide sequence of MHP55-2comprising a nuclear localization signal and lackingan intron.

SEQ ID NO:83 plant-optimized nucleotide sequence of MHP77 comprising anuclear localization signal and lackingan intron.

SEQ ID NO:84 is the HR1 of the MHP14 target site.

SEQ ID NO:85 is the HR2 of the MHP14 target site.

SEQ ID NO:86 is the HR1 of the MHP55 target site.

SEQ ID NO:87 is the HR2 of the MHP55 target site.

SEQ ID NO:88 is the HR1 of the MHP77 target site.

SEQ ID NO:89 is the HR2 of the MHP77 target site.

SEQ ID NO: 90 is the HR1 of the MHP1 target site.

SEQ ID NO:91: is the HR2 of the MHP1 target site.

SEQ ID NO:92 is the HR1 of the MHP32 target site.

SEQ ID NO:93 is the HR2 of the MHP32 target site.

SEQ ID NO:94 is the HR1 of the MHP42 target site.

SEQ ID NO:95 is the HR2 of the MHP42 target site.

SEQ ID NO:96 is the HR1 of the MHP67 target site.

SEQ ID NO:97 is the HR2 of the MHP67 target site.

SEQ ID NO:98 is the HR1 of the MHP98 target site.

SEQ ID NO:99 is the HR2 of the MHP98 target site.

SEQ ID NO:100 is the HR1 of the MHP107 target site.

SEQ ID NO:101 is the HR2 of the MHP107 target site.

SEQ ID NO:102 is the HR1 of the MHP115 target site.

SEQ ID NO:103 is the HR2 of the MHP115 target site.

SEQ ID NO:104 is the nucleotide sequence of the plasmid PHP44285 (MHP14and donor DNA).

SEQ ID NO:105 is the nucleotide sequence of the plasmid PHP44779(MHP14+and donor DNA).

SEQ ID NO:106 is the nucleotide sequence of the MHP14TS probe.

SEQ ID NO:107 is the nucleotide sequence of the MHPTS14TS_Forward_MGBprimer.

SEQ ID NO:108 is the nucleotide sequence of the MHPTS14TS_Reverse_MGBprimer.

SEQ ID NO:109 is the nucleotide sequence of the primer 146775 on genomicHR1 side.

SEQ ID NO:110 is the nucleotide sequence of the primer 146773 on vectorHR1 side.

SEQ ID NO:111 is the nucleotide sequence of the primer 146772 on genomicHR2 side.

SEQ ID NO:112 is the nucleotide sequence of the primer 146778 on vectorHR2 side.

SEQ ID NO:113 is the nucleotide sequence of the primer mopatF2.

SEQ ID NO:114 is the nucleotide sequence of the primer mopatR2.

SEQ ID NO:115 is the nucleotide sequence of the MHP55TS probe sequence.

SEQ ID NO:116 is the nucleotide sequence of the MHPTS55_Forward_MGBprimer.

SEQ ID NO:117 is the nucleotide sequence of the MHP55TS_Reverse_MGBprimer.

SEQ ID NO:118 is the nucleotide sequence of the MHP77TS probe.

SEQ ID NO:119 is the nucleotide sequence of the MHP77TS_Forward_MGBprimer.

SEQ ID NO:120 is the nucleotide sequence of the MHP77TS_Reverse_MGBprimer.

DETAILED DESCRIPTION OF THE INVENTION

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants; reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

In the context of this disclosure, a number of terms and abbreviationsare used. The following definitions are provided.

As used herein a “complex transgenic trait locus” (plural: “complextransgenic trait loci”) is a chromosomal segment within a genomic regionof interest that comprises at least two altered target sequences thatare genetically linked to each other and can also comprise one or morepolynucleotides of interest as described hereinbelow. Each of thealtered target sequences in the complex transgenic trait locusoriginates from a corresponding target sequence that was altered, forexample, by a mechanism involving a double-strand break within thetarget sequence that was induced by a double-strand break-inducing agentof the invention. In certain embodiments of the invention, the alteredtarget sequences comprise a transgene.

As used herein, a “genomic region of interest” is a segment of achromosome in the genome of a plant that is desirable for producing acomplex transgenic trait locus or the segment of a chromosome comprisinga complex transgenic trait locus that was produced by the methods of theinvention. The genomic region of interest can include, for example, oneor more polynucleotides of interest prior to producing a complextransgenic trait locus therein. Generally, a genomic region of interestof the present invention comprises a segment of chromosome that is 0-15cM.

The term “recognition sequence” or “recognition site” as used hereinrefers to a DNA sequence at which a double-strand break is induced inthe plant cell genome by a double-strand break inducing agent. The terms“recognition sequence” and “recognition site” are used interchangeablyherein.

The terms “target site”, “target sequence”, “target locus”, “genomictarget site”, “genomic target sequence”, and “genomic target locus” asused interchangeably herein refer to a polynucleotide sequence in thegenome of a plant cell that comprises a recognition sequence for adouble-strand break inducing agent.

An “artificial target sequence” is a target sequence that has beenintroduced into the genome of a plant. Such an artificial targetsequence can be identical in sequence to an endogenous or native targetsequence in the genome of a plant but be located in a different position(i.e., a non-endogenous or non-native position) in the genome of aplant.

The terms “endogenous target sequence” and “native target sequence” areused interchangeable herein to refer to a target sequence that isendogenous or native to the genome of a plant and is at the endogenousor native position of that target sequence in the genome of the plant.

An “altered target sequence” refers to a target sequence as disclosedherein that comprises at least one alteration of the invention whencompared to non-altered target sequence. Such “alterations” of theinvention include, for example: (i) replacement of at least onenucleotide, (ii) a deletion of at least one nucleotide, (iii) aninsertion of at least one nucleotide, or (iv) any combination of(i)-(iii).

The term “double-strand-break-inducing agent” as used herein refers toany nuclease which produces a double-strand break in the targetsequence. Producing the double-strand break in a target sequence orother DNA can be referred to herein as “cutting” or “cleaving” thetarget sequence or other DNA. In some embodiments of the invention, thedouble-strand-break-inducing agent has been engineered (or modified) tocut a specific endogenous target sequence, wherein the endogenous targetsequence prior to being cut by the engineereddouble-strand-break-inducing agent was not a sequence that would havebeen recognized by a native (non-engineered or non-modified)double-strand-break-inducing agent.

As used herein, “physically linked,” “in physical linkage”, and“genetically linked” are used to refer to any two or more genes,transgenes, native genes, mutated genes, alterations, target sites,markers, and the like that are part of the same DNA molecule orchromosome.

As used herein, a “polynucleotide of interest” within a genomic regionof interest is any coding and/or non-coding portion of the genomicregion of interest including, but not limited to, a transgene, a nativegene, a mutated gene, and a genetic marker such as, for example, asingle nucleotide polymorphism (SNP) marker and a simple sequence repeat(SSR) marker.

“Open reading frame” is abbreviated ORF.

As used herein, “nucleic acid” means a polynucleotide and includes asingle or a double-stranded polymer of deoxyribonucleotide orribonucleotide bases. Nucleic acids may also include fragments andmodified nucleotides. Thus, the terms “polynucleotide”, “nucleic acidsequence”, “nucleotide sequence” and “nucleic acid fragment” are usedinterchangeably to denote a polymer of RNA and/or DNA that is single- ordouble-stranded, optionally containing synthetic, non-natural, oraltered nucleotide bases. Nucleotides (usually found in their5′-monophosphate form) are referred to by their single letterdesignation as follows: “A” for adenosine or deoxyadenosine (for RNA orDNA, respectively), “C” for cytosine or deoxycytosine, “G” for guanosineor deoxyguanosine, “U” for uridine, “T” for deoxythymidine, “R” forpurines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” forA or C or T, “I” for inosine, and “N” for any nucleotide.

The terms “subfragment that is functionally equivalent” and“functionally equivalent subfragment” are used interchangeably herein.These terms refer to a portion or subsequence of an isolated nucleicacid fragment in which the ability to alter gene expression or produce acertain phenotype is retained whether or not the fragment or subfragmentencodes an active enzyme. For example, the fragment or subfragment canbe used in the design of chimeric genes to produce the desired phenotypein a transformed plant. Chimeric genes can be designed for use insuppression by linking a nucleic acid fragment or subfragment thereof,whether or not it encodes an active enzyme, in the sense or antisenseorientation relative to a plant promoter sequence.

The term “conserved domain” or “motif” means a set of amino acidsconserved at specific positions along an aligned sequence ofevolutionarily related proteins. While amino acids at other positionscan vary between homologous proteins, amino acids that are highlyconserved at specific positions indicate amino acids that are essentialto the structure, the stability, or the activity of a protein. Becausethey are identified by their high degree of conservation in alignedsequences of a family of protein homologues, they can be used asidentifiers, or “signatures”, to determine if a protein with a newlydetermined sequence belongs to a previously identified protein family.

Polynucleotide and polypeptide sequences, variants thereof, and thestructural relationships of these sequences can be described by theterms “homology”, “homologous”, “substantially identical”,“substantially similar” and “corresponding substantially” which are usedinterchangeably herein. These refer to polypeptide or nucleic acidfragments wherein changes in one or more amino acids or nucleotide basesdo not affect the function of the molecule, such as the ability tomediate gene expression or to produce a certain phenotype. These termsalso refer to modification(s) of nucleic acid fragments that do notsubstantially alter the functional properties of the resulting nucleicacid fragment relative to the initial, unmodified fragment. Thesemodifications include deletion, substitution, and/or insertion of one ormore nucleotides in the nucleic acid fragment.

Substantially similar nucleic acid sequences encompassed may be definedby their ability to hybridize (under moderately stringent conditions,e.g., 0.5X SSC, 0.1° A SDS, 60° C.) with the sequences exemplifiedherein, or to any portion of the nucleotide sequences disclosed hereinand which are functionally equivalent to any of the nucleic acidsequences disclosed herein. Stringency conditions can be adjusted toscreen for moderately similar fragments, such as homologous sequencesfrom distantly related organisms, to highly similar fragments, such asgenes that duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 80% sequence identity, or 90% sequence identity, upto and including 100% sequence identity (i.e., fully complementary) witheach other.

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will selectivelyhybridize to its target sequence in an in vitro hybridization assay.Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences can beidentified which are 100% complementary to the probe (homologousprobing). Alternatively, stringency conditions can be adjusted to allowsome mismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, optionally less than 500 nucleotides inlength.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salt(s)) at pH 7.0 to 8.3, and atleast about 30° C. for short probes (e.g., 10 to 50 nucleotides) and atleast about 60° C. for long probes (e.g., greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. Exemplary low stringencyconditions include hybridization with a buffer solution of 30 to 35%formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and awash in 1X to 2X SSC (20X SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50to 55° C. Exemplary moderate stringency conditions include hybridizationin 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5Xto 1X SSC at 55 to 60° C. Exemplary high stringency conditions includehybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a washin 0.1X SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth et al., (1984) Anal Biochem 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with 90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermalmelting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than thethermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). Hybridization and/or washconditions can be applied for at least 10, 30, 60, 90, 120 or 240minutes.

“Sequence identity” or “identity” in the context of nucleic acid orpolypeptide sequences refers to the nucleic acid bases or amino acidresidues in two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window.

The term “percentage of sequence identity” refers to the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) as compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the results by 100to yield the percentage of sequence identity. Useful examples of percentsequence identities include, but are not limited to, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% or any integer percentage from 50% to100%. These identities can be determined using any of the programsdescribed herein.

Sequence alignments and percent identity or similarity calculations maybe determined using a variety of comparison methods designed to detecthomologous sequences including, but not limited to, the MegAlign™program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.). Within the context of this application it will beunderstood that where sequence analysis software is used for analysis,that the results of the analysis will be based on the “default values”of the program referenced, unless otherwise specified. As used herein“default values” will mean any set of values or parameters thatoriginally load with the software when first initialized.

The “Clustal V method of alignment” corresponds to the alignment methodlabeled Clustal V (described by Higgins and Sharp, (1989) CABIOS5:151-153; Higgins et al., (1992) Comput Appl Biosci 8:189-191) andfound in the MegAlign™ program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). For multiple alignments, thedefault values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10.Default parameters for pairwise alignments and calculation of percentidentity of protein sequences using the Clustal method are KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids theseparameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.After alignment of the sequences using the Clustal V program, it ispossible to obtain a “percent identity” by viewing the “sequencedistances” table in the same program.

The “Clustal W method of alignment” corresponds to the alignment methodlabeled Clustal W (described by Higgins and Sharp, (1989) CABIOS5:151-153; Higgins et al., (1992) Comput Appl Biosci 8:189-191) andfound in the MegAlign™ v6.1 program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Default parameters formultiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, DelayDivergen Seqs (%)=30, DNA Transition Weight=0.5, Protein WeightMatrix=Gonnet Series, DNA Weight Matrix=IUB). After alignment of thesequences using the Clustal W program, it is possible to obtain a“percent identity” by viewing the “sequence distances” table in the sameprogram.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 (GCG, Accelrys,San Diego, Calif.) using the following parameters: % identity and %similarity for a nucleotide sequence using a gap creation penalty weightof 50 and a gap length extension penalty weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using a GAP creation penalty weight of 8 and a gap lengthextension penalty of 2, and the BLOSUM62 scoring matrix (Henikoff andHenikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915). GAP uses thealgorithm of Needleman and Wunsch, (1970) J Mol Biol 48:443-53, to findan alignment of two complete sequences that maximizes the number ofmatches and minimizes the number of gaps. GAP considers all possiblealignments and gap positions and creates the alignment with the largestnumber of matched bases and the fewest gaps, using a gap creationpenalty and a gap extension penalty in units of matched bases.

BLAST® is a searching algorithm provided by the National Center forBiotechnology Information (NCBI) used to find regions of similaritybetween biological sequences. The program compares nucleotide or proteinsequences to sequence databases and calculates the statisticalsignificance of matches to identify sequences having sufficientsimilarity to a query sequence such that the similarity would not bepredicted to have occurred randomly. BLAST® reports the identifiedsequences and their local alignment to the query sequence.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides from otherspecies or modified naturally or synthetically wherein such polypeptideshave the same or similar function or activity. Useful examples ofpercent identities include, but are not limited to, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% or any integer percentage from 50% to100%. Indeed, any integer amino acid identity from 50% to 100% may beuseful in describing the present invention, such as 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99%.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature, or at a different genetic locus than that found in nature. A“foreign” gene refers to a gene not normally found in the host organism,but that is introduced into the host organism by gene transfer. Foreigngenes can comprise native genes inserted into a non-native organism, orchimeric genes.

A “mutated gene” is a native gene that has been altered through humanintervention. Such a “mutated gene” has a sequence that differs from thesequence of the corresponding native gene by at least one nucleotideaddition, deletion, or substitution. In certain embodiments of theinvention, the mutated gene comprises an alteration that results from adouble-strand-break-inducing agent as disclosed herein.

A “transgene” is a gene that has been introduced into the genome by atransformation procedure. A transgene can, for example encode one ormore proteins or RNA that is not translated into protein. However, atransgene of the invention need not encode a protein and/ornon-translated RNA. In certain embodiments of the invention, thetransgene comprises one or more chimeric genes, including chimeric genescomprising, for example, a gene of interest, phenotypic marker, aselectable marker, and a DNA for gene silencing.

As used herein, a “targeted mutation” is mutation in a native gene thatwas made by altering a target sequence within the native gene using amethod involving a double-strand-break-inducing agent that is capable ofinducing a double-strand break in the DNA of the target sequence asdisclosed herein or known in the art.

The term “genome” as it applies to a plant cells encompasses not onlychromosomal DNA found within the nucleus, but organelle DNA found withinsubcellular components (e.g., mitochondria, or plastid) of the cell.

A “codon-modified gene” or “codon-preferred gene” or “codon-optimizedgene” is a gene having its frequency of codon usage designed to mimicthe frequency of preferred codon usage of the host cell. An “allele” isone of several alternative forms of a gene occupying a given locus on achromosome. When all the alleles present at a given locus on achromosome are the same, that plant is homozygous at that locus. If thealleles present at a given locus on a chromosome differ, that plant isheterozygous at that locus.

“Coding sequence” refers to a polynucleotide sequence which codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to: promoters, translation leader sequences, 5′untranslated sequences, 3′ untranslated sequences, introns,polyadenylation recognition sequences, RNA processing sites, effectorbinding sites, and stem-loop structures.

“A plant-optimized nucleotide sequence” is nucleotide sequence that hasbeen optimized for increased expression in plants, particularly forincreased expression in plants or in one or more plants of interest. Forexample, a plant-optimized nucleotide sequence can be synthesized bymodifying a nucleotide sequence encoding a protein such as, for example,double-strand-break-inducing agent (e.g., an endonuclease) as disclosedherein, using one or more plant-preferred codons for improvedexpression. See, for example, Campbell and Gowri (1990) Plant Physiol.92:1-11 for a discussion of host-preferred codon usage. Methods areavailable in the art for synthesizing plant-preferred genes. See, forexample, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al.(1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.Additional sequence modifications are known to enhance gene expressionin a plant host. These include, for example, elimination of: one or moresequences encoding spurious polyadenylation signals, one or moreexon-intron splice site signals, one or more transposon-like repeats,and other such well-characterized sequences that may be deleterious togene expression. The G-C content of the sequence may be adjusted tolevels average for a given plant host, as calculated by reference toknown genes expressed in the host plant cell. When possible, thesequence is modified to avoid one or more predicted hairpin secondarymRNA structures. Thus, “a plant-optimized nucleotide sequence” of thepresent invention comprises one or more of such sequence modifications.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. An “enhancer” is a DNA sequencethat can stimulate promoter activity, and may be an innate element ofthe promoter or a heterologous element inserted to enhance the level ortissue-specificity of a promoter. Promoters may be derived in theirentirety from a native gene, or be composed of different elementsderived from different promoters found in nature, and/or comprisesynthetic DNA segments. It is understood by those skilled in the artthat different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity. Promoters that cause agene to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”. New promoters of various typesuseful in plant cells are constantly being discovered; numerous examplesmay be found in the compilation by Okamuro and Goldberg, (1989) In TheBiochemistry of Plants, Vol. 115, Stumpf and Conn, eds (New York, NY:Academic Press), pp. 1-82.

“Translation leader sequence” refers to a polynucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (e.g., Turner and Foster, (1995) MolBiotechnol 3:225-236).

“3′ non-coding sequences”, “transcription terminator” or “terminationsequences” refer to DNA sequences located downstream of a codingsequence and include polyadenylation recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The use of different 3′ non-codingsequences is exemplified by Ingelbrecht et al., (1989) Plant Cell1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript. A RNA transcript is referred toas the mature RNA when it is a RNA sequence derived frompost-transcriptional processing of the primary transcript. “MessengerRNA” or “mRNA” refers to the RNA that is without introns and that can betranslated into protein by the cell. “cDNA” refers to a DNA that iscomplementary to, and synthesized from, a mRNA template using the enzymereverse transcriptase. The cDNA can be single-stranded or converted intodouble-stranded form using the Klenow fragment of DNA polymerase I.“Sense” RNA refers to RNA transcript that includes the mRNA and can betranslated into protein within a cell or in vitro. “Antisense RNA”refers to an RNA transcript that is complementary to all or part of atarget primary transcript or mRNA, and that blocks the expression of atarget gene (see, e.g., U.S. Pat. No. 5,107,065). The complementarity ofan antisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to antisense RNA, ribozymeRNA, or other RNA that may not be translated but yet has an effect oncellular processes. The terms “complement” and “reverse complement” areused interchangeably herein with respect to mRNA transcripts, and aremeant to define the antisense RNA of the message.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis regulated by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of regulating the expressionof that coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions can beoperably linked, either directly or indirectly, 5′ to the target mRNA,or 3′ to the target mRNA, or within the target mRNA, or a firstcomplementary region is 5′ and its complement is 3′ to the target mRNA.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal., Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y. (1989). Transformation methods arewell known to those skilled in the art and are described infra.

“PCR” or “polymerase chain reaction” is a technique for the synthesis ofspecific DNA segments and consists of a series of repetitivedenaturation, annealing, and extension cycles. Typically, adouble-stranded DNA is heat denatured, and two primers complementary tothe 3′ boundaries of the target segment are annealed to the DNA at lowtemperature, and then extended at an intermediate temperature. One setof these three consecutive steps is referred to as a “cycle”.

The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis,or manipulation of isolated segments of nucleic acids by geneticengineering techniques.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form ofdouble-stranded DNA. Such elements may be autonomously replicatingsequences, genome integrating sequences, phage, or nucleotide sequences,in linear or circular form, of a single- or double-stranded DNA or RNA,derived from any source, in which a number of nucleotide sequences havebeen joined or recombined into a unique construction which is capable ofintroducing a polynucleotide of interest into a cell. “Transformationcassette” refers to a specific vector containing a foreign gene andhaving elements in addition to the foreign gene that facilitatestransformation of a particular host cell. “Expression cassette” refersto a specific vector containing a foreign gene and having elements inaddition to the foreign gene that allow for expression of that gene in aforeign host.

The terms “recombinant DNA molecule”, “recombinant construct”,“expression construct”, “chimeric construct”, “construct”, and“recombinant DNA construct” are used interchangeably herein. Arecombinant construct comprises an artificial combination of nucleicacid fragments, e.g., regulatory and coding sequences that are not allfound together in nature. For example, a chimeric construct may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. Such a construct may be used by itself or may be used inconjunction with a vector. If a vector is used, then the choice ofvector is dependent upon the method that will be used to transform hostcells as is well known to those skilled in the art. For example, aplasmid vector can be used. The skilled artisan is well aware of thegenetic elements that must be present on the vector in order tosuccessfully transform, select and propagate host cells. The skilledartisan will also recognize that different independent transformationevents may result in different levels and patterns of expression (Joneset al., (1985) EMBO J 4:2411-2418; De Almeida et al., (1989) Mol GenGenetics 218:78-86), and thus that multiple events are typicallyscreened in order to obtain lines displaying the desired expressionlevel and pattern. Such screening may be accomplished standard molecularbiological, biochemical, and other assays including Southern analysis ofDNA, Northern analysis of mRNA expression, PCR, real time quantitativePCR (qPCR), reverse transcription PCR (RT-PCR), immunoblotting analysisof protein expression, enzyme or activity assays, and/or phenotypicanalysis.

The term “expression”, as used herein, refers to the production of afunctional end-product (e.g., an mRNA or a protein) in either precursoror mature form.

The term “introduced” means providing a nucleic acid (e.g., expressionconstruct) or protein into a cell. Introduced includes reference to theincorporation of a nucleic acid into a eukaryotic or prokaryotic cellwhere the nucleic acid may be incorporated into the genome of the cell,and includes reference to the transient provision of a nucleic acid orprotein to the cell. Introduced includes reference to stable ortransient transformation methods, as well as sexually crossing. Thus,“introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct/expression construct) into a cell, means“transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid fragment into aeukaryotic or prokaryotic cell where the nucleic acid fragment may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid, or mitochondrial DNA), converted into an autonomous replicon,or transiently expressed (e.g., transfected mRNA).

“Mature” protein refers to a post-translationally processed polypeptide(i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed). “Precursor” protein refers tothe primary product of translation of mRNA (i.e., with pre- andpropeptides still present). Pre- and propeptides may be but are notlimited to intracellular localization signals.

“Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance. Incontrast, “transient transformation” refers to the transfer of a nucleicacid fragment into the nucleus, or other DNA-containing organelle, of ahost organism resulting in gene expression without integration or stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms.

As used herein, “transgenic” refers to a plant or a cell which compriseswithin its genome a heterologous polynucleotide. Typically, theheterologous polynucleotide is stably integrated within the genome suchthat the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of an expression construct. Transgenic is used herein to includeany cell, cell line, callus, tissue, plant part or plant, the genotypeof which has been altered by the presence of heterologous nucleic acidincluding those transgenics initially so altered as well as thosecreated by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

The term “plant” refers to whole plants, plant organs, plant tissues,seeds, plant cells, seeds and progeny of the same. Plant cells include,without limitation, cells from seeds, suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen and microspores. Plant parts includedifferentiated and undifferentiated tissues including, but not limitedto roots, stems, shoots, leaves, pollens, seeds, tumor tissue andvarious forms of cells and culture (e.g., single cells, protoplasts,embryos, and callus tissue). The plant tissue may be in plant or in aplant organ, tissue or cell culture. The term “plant organ” refers toplant tissue or a group of tissues that constitute a morphologically andfunctionally distinct part of a plant. The term “genome” refers to theentire complement of genetic material (genes and non-coding sequences)that is present in each cell of an organism, or virus or organelle;and/or a complete set of chromosomes inherited as a (haploid) unit fromone parent. “Progeny” comprises any subsequent generation of a plant.

A “fertile plant” is a plant that is capable of producing a progenyplant. In certain embodiments of the invention, a fertile plant is aplant that produces viable male and female gametes and is self fertile.Such a self-fertile plant can produce a progeny plant without thecontribution from any other plant of a gamete and the genetic materialcontained therein. Other embodiments of the invention can involve theuse of a plant that is not self fertile because the plant does notproduce male or female gametes that are viable or otherwise capable offertilization. As used herein, a “male sterile plant” is a plant thatdoes not produce male gametes that are viable or otherwise capable offertilization. As used herein, a “female sterile plant” is a plant thatdoes not produce female gametes that are viable or otherwise capable offertilization. It is recognized that male and female sterile plants canbe female and mail fertile, respectively. It is further recognized thata male fertile (but female sterile) plant can produce viable progenywhen crossed with a female fertile plant and that a female fertile (butmale sterile) plant can produce viable progeny when crossed with a malefertile plant.

A “centimorgan” (cM) or “map unit” is the distance between two linkedgenes, markers, target sites, loci, or any pair thereof, wherein 1% ofthe products of meiosis are recombinant. Thus, a centimorgan isequivalent to a distance equal to an 1% average recombination frequencybetween the two linked genes, markers, target sites, loci, or any pairthereof.

The present invention finds use in the breeding of plants comprising twoto more transgenic traits. Currently, transgenic traits are randomlyinserted throughout the plant genome as a consequence of transformationsystems based on Agrobacterium, biolistics, or other commonly usedprocedures. More recently, gene targeting protocols have been developedthat enable directed transgene insertion. One important technology,site-specific integration (SSI) enables the targeting of a transgene tothe same chromosomal location as a previously inserted transgene.Custom-designed meganucleases and custom-designed zinc fingermeganucleases allow researchers to design nucleases to target specificchromosomal locations, and these reagents allow the targeting oftransgenes at the chromosomal site cleaved by these nucleases.

As disclosed herein, nuclease-mediated gene targeting can be used inmethods for producing complex transgenic trait loci comprising multipletransgenes. In one embodiment of the invention, a complex transgenictrait locus is a locus that has multiple transgenes genetically linkedto each other. By inserting independent transgenes within 1, 2 or even 5centimorgans (cM) from each other, the transgenes can be bred as singlegenetic locus. FIG. 7 depicts the process of how two traits could beintegrated into the genome at a genetic distance of, for example, 0.2 cMfrom each other in independent transformation runs or in sequentialtransformations (e.g., transformation and re-transformation). Afterselecting the events, plants containing the two events can be crossed toform an F1 that contains the events on different chromosomes. In progenyfrom these F1 (F2 or BC1) 1/500 progeny would have the two differenttransgenes recombined onto the same chromosome. The complex locus couldthen be bred as single genetic locus with both transgene traits. Thisprocess could be repeated to stack as many traits as desired.

The present invention provides methods for producing complex transgenictrait loci at selected genomic regions to simplify breeding withmultiple transgenes. To initiate the development of a complex transgenictrait locus, a region of the genome is first selected. Second, thesequence of nearby genomic regions is compiled and nuclease reagentsdesigned to facilitate targeting additional transgenes to those closelylinked sites. Subsequently, algorithms for nuclease design such as, forexample, those described in U.S. Patent Application Publication No.2007/0117128 A1 are used to select potential target sites. Additionalbioinformatic analysis such as, for example, copy number of the site inthe target genome, location of the site relative to known gene codingregions and other factors could be used to filter the sites to a subsetof preferred sites. Nucleases could then be used to target newtransgenes to these preferred sites using published protocols See, forexample, Halluin et al. (2008) Plant Biotechnol. J. 6:93-102; Shukla etal. (2009) Nature doi:10.1038/nature07992; Wright et al. Plant J. (2005)44:693-705; and WO 2009/006297); all of which are herein incorporated byreference.

In a first aspect, the present invention provides methods for producingin a plant a complex transgenic trait locus comprising at least twoaltered target sequences in a genomic region of interest. In oneembodiment, the methods involve selecting a genomic region in a plantthat comprises a first target sequence and a second target sequence.Generally, the first target sequence and the second target sequence areseparated from each other by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8., 0.9, 1, 2, 3, 4, or 5 centimorgans (cM) in the genome of theplant. In certain embodiments of the invention, the first and secondtarget sequences are physically linked to a polynucleotide of interestsuch as, for example, a transgene, native gene, or a gene with atargeted mutation, that is within about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8., 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or 21 cM of the first and/or the second target sequence.

The methods of the invention further involve providing a firstdouble-strand-break-inducing agent and a seconddouble-strand-break-inducing agent. The firstdouble-strand-break-inducing agent is capable of inducing a firstdouble-strand break in DNA comprising the first target sequence, and thesecond double-strand-break-inducing agent is capable of inducing asecond double-strand break in DNA comprising the second target sequence.The methods of the invention do not depend on a particulardouble-strand-break-inducing agent but only that thedouble-strand-break-inducing agent is capable of inducing adouble-strand break in DNA in a target sequence of the invention. Anysuch double-strand-break-inducing agent that is disclosed herein orknown in the art can be used in the methods of the present invention.

Additionally, the methods involve contacting at least one plant cellwith the first double-strand-break-inducing agent, identifying a cellcomprising a first alteration at the first target sequence, and thenrecovering a first fertile plant from the cell comprising the firstalteration. The first fertile plant also comprises the first alteration.Additionally, the method involves contacting at least one plant cellwith the second double-strand-break-inducing agent, identifying a cellcomprising a second alteration at the second target sequence, and thenrecovering a second fertile plant from the cell comprising the secondalteration. The method further involves obtaining a fertile progenyplant from the second fertile plant, wherein the fertile progeny plantcomprises both the first and second alterations in physical linkage.

In one embodiment of this method, the fertile progeny plant is obtainedby crossing the first fertile plant and the second fertile plant andselecting for a fertile progeny plant comprising both the first andsecond alterations in physical linkage. In another embodiment, a cell ofthe first fertile plant, or progeny thereof comprising the firstalteration, is contacted with the second double-strand-break-inducingagent, and the second fertile plant comprises both the first and secondalterations, which may or may not be physically linked. If necessary,the second fertile plant can be selfed and a fertile progeny plantselected comprising both the first and second alterations in physicallinkage.

The first and second alterations are selected from the group consistingof (i) replacement of at least one nucleotide, (ii) a deletion of atleast one nucleotide, (iii) an insertion of at least one nucleotide, and(iv) any combination of (i)-(iii). In one embodiment of the invention,the first and/or the second alterations comprise insertion of a DNAsequence of interest including, but not limited to, a DNA for genesilencing, a DNA encoding a phenotypic marker, and a DNA encoding aprotein providing an agronomic advantage. In another embodiment, thefirst and/or the second alterations comprise a targeted mutation in anative gene.

In a like manner, the methods disclosed herein can be used to produce ina plant a complex transgenic trait locus comprising 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more altered target sequences in physical linkage in agenomic region of interest comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore target sequences of interest. Each additional target sequence ofinterest in the genomic region of interest can be recognized and cleavedby a double-strand-break-inducing agent essentially as described above.

For example, a third DNA sequence of interest is inserted into a thirdtarget sequence by contacting at least one cell of a plant with a thirddouble-strand-break-inducing agent and a third DNA molecule comprisingthe DNA sequence of interest, and then identifying a cell comprising theDNA sequence of interest. The method can further comprising recovering afertile plant comprising the third DNA sequence of interest. In oneembodiment, the cell comprising the third DNA sequence of interestcomprises the first alteration, the second alteration, or both the firstalteration and the second alteration. The method of the invention canfurther comprising producing a fertile plant comprising the firstalteration, the second alteration, and the third DNA sequence ofinterest in physical linkage. In another embodiment, the fertile plantcomprising the first alteration, the second alteration, and the thirdDNA sequence of interest is produced by crossing the fertile plantcomprising the first and second alterations with a second fertile plantcomprising the third DNA sequence of interest, and selecting a fertileprogeny plant from the crossing, wherein the fertile progeny plantcomprises the first alteration, the second alteration, and the third DNAsequence of interest in physical linkage.

The fertile plant comprising the first alteration, the secondalteration, and the third DNA sequence of interest can be produced, forexample, by: (i) contacting a cell comprising the first alteration andthe second alteration with the third double-strand-break-inducing agent;(ii) identifying a cell from (i) comprising the third DNA sequence ofinterest, wherein the cell comprises the first alteration and the secondalteration, and wherein the first alteration, the second alteration, andthe third DNA sequence of interest are physically linked; and (iii)recovering a fertile plant comprising in physical linkage the firstalteration, the second alteration, and the third DNA sequence ofinterest.

In another embodiment of the invention, the methods for producing in aplant a complex transgenic trait locus comprising at least two alteredtarget sequences in a genomic region of interest that involve obtaininga first fertile plant comprising a first altered target site at thegenomic region of interest and a second fertile plant comprising asecond altered target site at the genomic region of interest. In thismethod, the first altered target sequence originated from a first targetsequence that is recognized and cleaved by a firstdouble-strand-break-inducing agent, and the second altered targetsequence originated from a second target sequence that is recognized andcleaved by a second double-strand-break-inducing agent. The secondmethod further involves crossing the first fertile plant and the secondfertile plant, and then selecting from the crossing a fertile progenyplant comprising the first alteration and the second alteration inphysical linkage.

The second method can optionally involve crossing the fertile progenyplant with an additional fertile plant that comprises at least a thirdaltered target sequence in the genomic region of interest and thenselecting from the crossing a fertile progeny plant comprising the firstalteration, the second and the at least third alteration in physicallinkage. Like the first and second altered target sequences, the thirdaltered target sequence originated from a third target sequence that isrecognized and cleaved by a third double-strand-break-inducing agent. Ina like manner, a complex transgenic trait locus can be producedcomprising 4, 5, 6, 7, 8, 9, 10, or more altered target sequences inphysical linkage in the genomic region of interest.

In another aspect, the present invention provides complex transgenictrait loci in plants as well as plants, plant parts, plant cells, andseeds comprising at least one complex transgenic trait locus of theinvention. A complex transgenic trait locus of the invention comprisesat least two altered target sequences that are genetically linked to apolynucleotide of interest. Such altered target sequences originatedfrom a corresponding target sequence that is recognized and cleaved by adouble-strand-break-inducing agent using, for example, the methodsdisclosed herein. The altered target sequences comprise an alterationsuch as, for example, replacement of at least one nucleotide in thetarget sequence, a deletion of at least one nucleotide in the targetsequence, an insertion of at least one nucleotide in the targetsequence, or any combination thereof. The polynucleotide interest canbe, for example, a transgene, a native gene, and a mutated gene. Thepresent invention provides plants, plant parts, plant cells, and seedscomprising at least one complex transgenic trait locus of the invention.

In one embodiment, a complex transgenic trait locus of the inventioncomprises at least one altered target sequence comprising a recombinantDNA molecule. Recombinant DNA molecules of the invention include, butare not limited to, a DNA for gene silencing, a DNA encoding aphenotypic marker, and a DNA encoding a protein providing an agronomicadvantage.

Generally, each of the altered target sites of the complex transgenictrait locus are located within about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8., 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or 21 centimorgan (cM) of the polynucleotide of interest.

The methods of the present invention involve the use of one or moredouble-strand break inducing agents. A double-strand break inducingagent of the present invention is any agent that recognizes and/or bindsto a specific polynucleotide recognition sequence to produce a break inthe target sequence at or near the recognition sequence. Examples ofdouble-strand break inducing agents include, but are not limited to,endonucleases, TAL effector nucleases, and zinc finger nucleases, andinclude modified derivatives, variants, and fragments thereof.

A recognition sequence is any polynucleotide sequence that isspecifically recognized and/or bound by a double-strand break inducingagent. The length of the recognition site sequence can vary, andincludes, for example, sequences that are at least 4, 6, 8, 10, 12, 14,16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70or more nucleotides in length.

It is possible that the recognition site could be palindromic, that is,the sequence on one strand reads the same in the opposite direction onthe complementary strand. The nick/cleavage site could be within therecognition sequence or the nick/cleavage site could be outside of therecognition sequence. In another variation, the cleavage could occur atnucleotide positions immediately opposite each other to produce a bluntend cut or, in other cases, the incisions could be staggered to producesingle-stranded overhangs, also called “sticky ends”, which can beeither 5′ overhangs, or 3′ overhangs. The recognition sequence can beendogenous or exogenous. When the recognition site is an endogenoussequence, it may be a recognition sequence recognized by anaturally-occurring, or native double-strand break inducing agent.Alternatively, an endogenous recognition site could be recognized and/orbound by a modified or engineered double-strand break inducing agentdesigned or selected to specifically recognize the endogenousrecognition sequence to produce a double-strand break. A modifieddouble-strand break inducing agent can be derived from a native,naturally-occurring double-strand break inducing agent or it could beartificially created or synthesized.

A variety of methods are available to identify those cells having analtered genome at or near the recognition sequence without using ascreenable marker phenotype. Such methods can be viewed as directlyanalyzing a recognition sequence to detect any change in the recognitionsequence, including but not limited to PCR methods, sequencing methods,nuclease digestion, Southern blots, and any combination thereof.

Proteins may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known. For example, amino acid sequencevariants of the protein(s) can be prepared by mutations in the DNA.Methods for mutagenesis and nucleotide sequence alterations include, forexample, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-92; Kunkel etal., (1987) Meth Enzymol 154:367-82; U.S. Pat. No. 4,873,192; Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York) and the references cited therein. Guidanceregarding amino acid substitutions not likely to affect biologicalactivity of the protein is found, for example, in the model of Dayhoffet al., (1978) Atlas of Protein Sequence and Structure (Natl Biomed ResFound, Washington, D.C.). Conservative substitutions, such as exchangingone amino acid with another having similar properties, may bepreferable. Conservative deletions, insertions, and amino acidsubstitutions are not expected to produce radical changes in thecharacteristics of the protein, and the effect of any substitution,deletion, insertion, or combination thereof can be evaluated by routinescreening assays. Assays for double-strand-break-inducing activity areknown and generally measure the overall activity and specificity of theagent on DNA substrates containing recognition sites.

Endonucleases are enzymes that cleave the phosphodiester bond within apolynucleotide chain, and include restriction endonucleases that cleaveDNA as specific sites without damaging the bases. Restrictionendonucleases include Type I, Type II, Type III, and Type IVendonucleases, which further include subtypes. In the Type I and TypeIII systems, both the methylase and restriction activities are containedin a single complex.

Type I and Type III restriction endonucleases recognize specificrecognition sites, but typically cleave at a variable position from therecognition site, which can be hundreds of base pairs away from therecognition site. In Type II systems the restriction activity isindependent of any methylase activity, and cleavage typically occurs atspecific sites within or near to the recognition site. Most Type IIenzymes cut palindromic sequences, however Type IIa enzymes recognizenon-palindromic recognition sites and cleave outside of the recognitionsite, Type IIb enzymes cut sequences twice with both sites outside ofthe recognition site, and Type IIs enzymes recognize an asymmetricrecognition site and cleave on one side and at a defined distance ofabout 1-20 nucleotides from the recognition site.

Type IV restriction enzymes target methylated DNA. Restriction enzymesare further described and classified, for example in the REBASE database(webpage at rebase.neb.com; Roberts et al., (2003) Nucleic Acids Res31:418-20), Roberts et al., (2003) Nucleic Acids Res 31:1805-12, andBelfort et al., (2002) in Mobile DNA II, pp. 761-783, Eds. Craigie etal., (ASM Press, Washington, DC).

Endonucleases also include meganucleases, also known as homingendonucleases (HEases), which like restriction endonucleases, bind andcut at a specific recognition sequence, however the recognition sitesfor meganucleases are typically longer, about 18 bp or more.Meganucleases have been classified into four families based on conservedsequence motifs, the families are the LAGLIDADG, GIY-YIG, H-N-H, andHis-Cys box families. These motifs participate in the coordination ofmetal ions and hydrolysis of phosphodiester bonds. HEases are notablefor their long recognition sites, and for tolerating some sequencepolymorphisms in their DNA substrates. The naming convention formeganuclease is similar to the convention for other restrictionendonuclease. Meganucleases are also characterized by prefix F-, I-, orPI- for enzymes encoded by free-standing ORFs, introns, and inteins,respectively. For example, intron-, intein-, and freestanding geneencoded meganuclease from Saccharomyces cerevisiae are denoted I-SceI,PI-SceI, and F-SceII, respectively. Meganuclease domains, structure andfunction are known, see for example, Guhan and Muniyappa (2003) Crit RevBiochem Mol Biol 38:199-248; Lucas et al., (2001) Nucleic Acids Res29:960-9; Jurica and Stoddard, (1999) Cell Mol Life Sci 55:1304-26;Stoddard, (2006) Q Rev Biophys 38:49-95; and Moure et al., (2002) NatStruct Biol 9:764. In some examples a naturally occurring variant,and/or engineered derivative meganuclease is used. Methods for modifyingthe kinetics, cofactor interactions, expression, optimal conditions,and/or recognition site specificity, and screening for activity areknown, see for example, Epinat et al., (2003) Nucleic Acids Res31:2952-62; Chevalier et al., (2002) Mol Cell 10:895-905; Gimble et al.,(2003) Mol Biol 334:993-1008; Seligman et al., (2002) Nucleic Acids Res30:3870-9; Sussman et al., (2004) J Mol Biol 342:31-41; Rosen et al.,(2006) Nucleic Acids Res 34:4791-800; Chames et al., (2005) NucleicAcids Res 33:e178; Smith et al., (2006) Nucleic Acids Res 34:e149; Gruenet al., (2002) Nucleic Acids Res 30:e29; Chen and Zhao, (2005) NucleicAcids Res 33:e154; WO2005105989; WO2003078619; WO2006097854;WO2006097853; WO2006097784; and WO2004031346.

Any meganuclease can be used as a double-strand break inducing agentincluding, but not limited to, I-SceI, I-SceII, I-SceIII, I-SceIV,I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAIIP, I-Crel, I-CrepsbIP,I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI,F-SceII, F-SuvI, F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI,I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI,I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI,I-NcIIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP,I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorI, I-PorIIP, I-PbpIP,I-SpBetaIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP,I-SpomIIP, I-SquIP, I-Ssp68031, I-SthPhiJP, I-SthPhiST3P,I-SthPhiSTe3bP, I-TdeIP, I-TevI, II-TevII, I-TevIII, I-UarAP,I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIPPI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP,PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, PI-TliII, orany variant or derivative thereof.

The endonuclease can be a modified endonuclease that binds a non-nativeor exogenous recognition sequence and does not bind a native orendogenous recognition sequence. Modification of the endonuclease can beas little as one nucleotide. A modified endonuclease is not capable ofmaking a double-strand break within a wild-type target sequence. Awild-type (i.e., prior to being modified) endonuclease is capable ofmaking a double-strand break within the wild-type target sequence.

The endonuclease can be provided via a polynucleotide encoding theendonuclease. Such a polynucleotide encoding an endonuclease can bemodified to substitute codons having a higher frequency of usage in aplant, as compared to the naturally occurring polynucleotide sequence.For example the polynucleotide encoding the endonuclease can be modifiedto substitute codons having a higher frequency of usage in a maize orsoybean plant, as compared to the naturally occurring polynucleotidesequence.

A site-specific recombinase, also referred to as a recombinase, is apolypeptide that catalyzes conservative site-specific recombinationbetween its compatible recombination sites, and includes nativepolypeptides as well as derivatives, variants and/or fragments thatretain activity, and native polynucleotides, derivatives, variants,and/or fragments that encode a recombinase that retains activity.

One step in the recombination process involves polynucleotide cleavageat or near the recognition site. This cleaving activity can be used toproduce a double-strand break. For reviews of site-specific recombinasesand their recognition sites, see, Sauer (1994) Curr Op Biotechnol5:521-7; and Sadowski (1993) FASEB 7:760-7. In some examples therecombinase is from the Integrase or Resolvase families.

The Integrase family of recombinases has over one hundred members andincludes, for example, FLP, Cre, lambda integrase, and R. The Integrasefamily has been grouped into two classes based on the structure of theactive sites, serine recombinases and tyrosine recombinases. Thetyrosine family, which includes Cre, FLP, SSV1, and lambda (λ)integrase, uses the catalytic tyrosine's hydroxyl group for anucleophilic attack on the phosphodiester bond of the DNA. Typically,members of the tyrosine family initially nick the DNA, which later formsa double-strand break. In the serine recombinase family, which includesphiC31 (ΦC31) integrase, a conserved serine residue forms a covalentlink to the DNA target site (Grindley et al., (2006) Ann Rev Biochem16:16). For other members of the Integrase family, see for example,Esposito et al., (1997) Nucleic Acids Res 25:3605-14 and Abremski etal., (1992) Protein Eng 5:87-91.

Other recombination systems include, for example, the streptomycetebacteriophage phiC31 (Kuhstoss et al., (1991) J Mol Biol 20:897-908);the SSV1 site-specific recombination system from Sulfolobus shibatae(Maskhelishvili et al., (1993) Mol Gen Genet 237:334-42); and aretroviral integrase-based integration system (Tanaka et al., (1998)Gene 17:67-76).

Sometimes the recombinase is one that does not require cofactors or asupercoiled substrate, including but not limited to Cre, FLP, and activederivatives, variants or fragments thereof. FLP recombinase catalyzes asite-specific reaction during DNA replication and amplification of thetwo-micron plasmid of S. cerevisiae. FLP recombinase catalyzessite-specific recombination between two FRT sites. The FLP protein hasbeen cloned and expressed (Cox (1993) Proc. Natl. Acad. Sci. USA80:4223-7). Functional derivatives, variants, and fragments of FLP areknown (Buchholz et al., (1998) Nat Biotechnol 16:617-8, Hartung et al.,(1998) J Biol Chem 273:22884-91, Saxena et al., (1997) Biochim BiophysActa 1340:187-204, and Hartley et al., (1980) Nature 286:860-4).

The bacteriophage recombinase Cre catalyzes site-specific recombinationbetween two lox sites (Guo et al., (1997) Nature 389:40-6; Abremski etal., (1984) J Biol Chem 259:1509-14; Chen et al., (1996) Somat Cell MolGenet 22:477-88; Shaikh et al., (1977) J Biol Chem 272:5695-702; andBuchholz et al., (1998) Nat Biotechnol 16:617-8). Examples ofsite-specific recombinases that can be used to produce a double-strandbreak at a recognition sequence, including for example FLP, Cre, SSV1,lambda Int, phi C31, HK022, and R. Examples of site-specificrecombination systems used in plants can be found in U.S. Pat. Nos.5,929,301; 6,175,056; WO99/25821; U.S. Pat. No. 6,331,661; WO99/25855;WO99/25841, and WO99/25840, the contents of each are herein incorporatedby reference.

Methods for modifying the kinetics, cofactor interaction andrequirements, expression, optimal conditions, and/or recognition sitespecificity, and screening for activity of recombinases and variants areknown, see for example Miller et al., (1980) Cell 20:721-9;Lange-Gustafson and Nash, (1984) J Biol Chem 259:12724-32; Christ etal., (1998) J Mol Biol 288:825-36; Lorbach et al., (2000) J Mol Biol296:1175-81; Vergunst et al., (2000) Science 290:979-82; Dorgai et al.,(1995) J Mol Biol 252:178-88; Dorgai et al., (1998) J Mol Biol277:1059-70; Yagu et al., (1995) J Mol Biol 252:163-7; Sclimente et al.,(2001) Nucleic Acids Res 29:5044-51; Santoro and Schultze, (2002) Proc.Natl. Acad. Sci. USA 99:4185-90; Buchholz and Stewart, (2001) NatBiotechnol 19:1047-52; Voziyanov et al., (2002) Nucleic Acids Res30:1656-63; Voziyanov et al., (2003) J Mol Biol 326:65-76; Klippel etal., (1988) EMBO J 7:3983-9; Arnold et al., (1999) EMBO J 18:1407-14;WO03/08045; WO99/25840; and WO99/25841. The recognition sites range fromabout 30 nucleotide minimal sites to a few hundred nucleotides.

Any recognition site for a recombinase can be used, including naturallyoccurring sites, and variants. Variant recognition sites are known, seefor example Hoess et al., (1986) Nucleic Acids Res 14:2287-300; Albertet al., (1995) Plant J 7:649-59; Thomson et al., (2003) Genesis36:162-7; Huang et al., (1991) Nucleic Acids Res 19:443-8; Siebler andBode, (1997) Biochemistry 36:1740-7; Schlake and Bode, (1994)Biochemistry 33:12746-51; Thygarajan et al., (2001) Mol Cell Biol21:3926-34; Umlauf and Cox, (1988) EMBO J 7:1845-52; Lee and Saito,(1998) Gene 216:55-65; WO01/23545; WO99/25821; WO99/25851; WO01/11058;WO01/07572 and U.S. Pat. No. 5,888,732.

A recombinase can be provided via a polynucleotide that encodes therecombinase or it can be provided via a modified polynucleotide encodingthe recombinase. For example, the polynucleotide (encoding arecombinase) can be modified to substitute codons having a higherfrequency of usage in a plant, as compared to the naturally occurringpolynucleotide sequence or it can be modified to substitute codonshaving a higher frequency of usage in a maize or soybean plant, ascompared to the naturally occurring polynucleotide sequence.

TAL effector nucleases are a new class of sequence-specific nucleasesthat can be used to make double-strand breaks at specific targetsequenes in the genome of a plant or other organism. TAL effectornucleases are created by fusing a native or engineered transcriptionactivator-like (TAL) effector, or functional part thereof, to thecatalytic domain of an endonuclease, such as, for example, FokI. Theunique, modular TAL effector DNA binding domain allows for the design ofproteins with potentially any given DNA recognition specificity. Thus,the DNA binding domains of the TAL effector nucleases can be engineeredto recognize specific DNA target sites and thus, used to makedouble-strand breaks at desired target sequences. See, WO 2010/079430;Morbitzer et al. (2010) PNAS 10.1073/pnas.1013133107; Scholze & Boch(2010) Virulence 1:428-432; Christian et al. Genetics (2010)186:757-761; Li et al. (2010) Nuc. Acids Res. (2010)doi:10.1093/nar/gkq704; and Miller et al. (2011) Nature Biotechnology29:143-148; all of which are herein incorporated by reference.

Transposases are polypeptides that mediate transposition of a transposonfrom one location in the genome to another. Transposases typicallyinduce double-strand breaks to excise the transposon, recognizesubterminal repeats, and bring together the ends of the excisedtransposon, in some systems other proteins are also required to bringtogether the ends during transposition.

Examples of transposons and transposases include, but are not limitedto, the Ac/Ds, Dt/rdt, Mu-M1/Mn, and Spm(En)/dSpm elements from maize,the Tam elements from snapdragon, the Mu transposon from bacteriophage,bacterial transposons (Tn) and insertion sequences (IS), Ty elements ofyeast (retrotransposon), Ta1 elements from Arabidopsis(retrotransposon), the P element transposon from Drosophila (Gloor etal., (1991) Science 253:1110-1117), the Copia, Mariner and Minoselements from Drosophila, the Hermes elements from the housefly, thePiggyBack elements from Trichplusia ni, Tc1 elements from C. elegans,and IAP elements from mice (retrotransposon). In some examples thetransposase is provided via a polynucleotide that encodes thetransposase.

It is possible to modify the polynucleotide encoding the transposase bysubstituting codons having a higher frequency of usage in a plant, ascompared to the naturally occurring polynucleotide sequence of bysubstituting codons having a higher frequency of usage in a maize orsoybean plant, as compared to the naturally occurring polynucleotidesequence.

DNA topoisomerases modulate DNA secondary and higher order structuresand functions related primarily to replication, transcription,recombination and repair. Topoisomerases share two characteristics: (i)the ability to cleave and reseal the phosphodiester backbone of DNA intwo successive transesterification reactions; and (ii) once atopoisomerase cleaved DNA intermediate is formed, the enzyme allows thesevered DNA ends to come apart, allowing the passage of another single-or double-stranded DNA segment. DNA topoisomerases can be classifiedinto three evolutionary independent families: type IA, type IB and typeII.

Those that cleave one strand of DNA and allow single step changes in thelinking number of circular DNA are defined as type I DNA topoisomerases.The Escherichia coli topoisomerase I and topoisomerase III,Saccharomyces cerevisiae topoisomerase III and reverse gyrase belong tothe type IA or type I-5′ subfamily as the protein link is to a 5′phosphate in the DNA. The prototype of type IB or I-3′ enzymes are foundin all eukaryotes and also in vaccinia virus topoisomerase I where theprotein is attached to a 3′ phosphate. Despite differences in mechanismand specificity between the bacterial and eukaryotic enzymes, yeast DNAtopoisomerase I can complement a bacterial DNA topoisomerase I mutant(Bjornsti et al., (1987) Proc. Natl. Acad. Sci. USA 84:8971-5). Type IAtopoisomerases relax negatively supercoiled DNA and require magnesiumand a single-stranded region of DNA. Topoisomerases IB relax bothpositively and negatively supercoiled DNA with equal efficiency and donot require a single-stranded region of DNA or metal ions for function.

The type II family includes E. coli DNA gyrase, E. coli topoisomerase IV(par E), eukaryotic type II topoisomerases, and archaic topoisomeraseVI. Type II enzymes are homodimeric (eukaryotic topoisomerase II) ortetrameric (gyrase), cleaving both strands of a duplex. Preferredcutting sites are known for available topoisomerases.

Zinc finger nucleases (ZFNs) are engineered double-strand break inducingagents comprised of a zinc finger DNA binding domain and adouble-strand-break-inducing agent domain. Recognition site specificityis conferred by the zinc finger domain, which typically comprising two,three, or four zinc fingers, for example having a C2H2 structure,however other zinc finger structures are known and have been engineered.Zinc finger domains are amenable for designing polypeptides whichspecifically bind a selected polynucleotide recognition sequence. ZFNsconsist of an engineered DNA-binding zinc finger domain linked to anon-specific endonuclease domain, for example nuclease domain from aType IIs endonuclease such as FokI. Additional functionalities can befused to the zinc-finger binding domain, including transcriptionalactivator domains, transcription repressor domains, and methylases. Insome examples, dimerization of nuclease domain is required for cleavageactivity. Each zinc finger recognizes three consecutive base pairs inthe target DNA. For example, a 3 finger domain recognized a sequence of9 contiguous nucleotides, with a dimerization requirement of thenuclease, two sets of zinc finger triplets are used to bind a 18nucleotide recognition sequence. A recognition sequence of 18nucleotides is long enough to be unique in a mammalian genome(4¹⁸=6.9×10¹⁰).

To date, designer zinc finger modules predominantly recognize GNN andANN triplets (Dreier et al., (2001) J Biol Chem 276:29466-78; Dreier etal., (2000) J Mol Biol 303:489-502; Liu et al., (2002) J Biol Chem277:3850-6), but examples using CNN or TNN triplets are also known(Dreier et al., (2005) J Biol Chem 280:35588-97; Jamieson et al., (2003)Nature Rev Drug Discov 2:361-8). See also, Durai et al., (2005) NucleicAcids Res 33:5978-90; Segal, (2002) Methods 26:76-83; Porteus andCarroll, (2005) Nat Biotechnol 23:967-73; zinc-finger consortium(website at www.zincfinger.org); Pabo et al., (2001) Ann Rev Biochem70:313-40; Wolfe et al., (2000) Ann Rev Biophys Biomol Struct29:183-212; Segal and Barbas, (2001) Curr Opin Biotechnol 12:632-7;Segal et al., (2003) Biochemistry 42:2137-48; Beerli and Barbas, (2002)Nat Biotechnol 20:135-41; Carroll et al., (2006) Nature Protocols1:1329; Ordiz et al., (2002) Proc. Natl. Acad. Sci. USA 99:13290-5; Guanet al., (2002) Proc. Natl. Acad. Sci. USA 99:13296-301; WO2002099084;WO00/42219; WO02/42459; WO2003062455; U.S. Patent ApplicationPublication No. 20030059767; U.S. Patent Application Publication No.2003/0108880; U.S. Pat. Nos. 6,140,466, 6,511,808 and 6,453,242.

Alternatively, engineered zinc finger DNA binding domains can be fusedto other double-strand break inducing agents or derivatives thereof thatretain DNA nicking/cleaving activity. For example, this type of fusioncan be used to direct the double-strand break inducing agent to adifferent target site, to alter the location of the nick or cleavagesite, to direct the inducing agent to a shorter target site, or todirect the inducing agent to a longer target site. In some examples azinc finger DNA binding domain is fused to a site-specific recombinase,transposase, topoisomerase, or a derivative thereof that retains DNAnicking and/or cleaving activity.

It is possible to provide a zinc-finger nuclease via a polynucleotidethat encodes the zinc-finger nuclease. This polynucleotide encoding thezinc-finger nuclease can be modified by substituting codons having ahigher frequency of usage in a plant, as compared to the naturallyoccurring polynucleotide sequence or by substituting codons having ahigher frequency of usage in a maize or soybean plant, as compared tothe naturally occurring polynucleotide sequence.

Sufficient homology or sequence identity indicates that twopolynucleotide sequences have sufficient structural similarity to act assubstrates for a homologous recombination reaction. The structuralsimilarity includes overall length of each polynucleotide fragment, aswell as the sequence similarity of the polynucleotides. Sequencesimilarity can be described by the percent sequence identity over thewhole length of the sequences, and/or by conserved regions comprisinglocalized similarities such as contiguous nucleotides having 100%sequence identity, and percent sequence identity over a portion of thelength of the sequences.

The amount of homology or sequence identity shared by a target and adonor polynucleotide can vary and includes total lengths and/or regionshaving unit integral values in the ranges of about 1-20 bp, 20-50 bp,50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp,300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb,2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including thetotal length of the target site. These ranges include every integerwithin the range, for example, the range of 1-20 bp includes 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bp. Theamount of homology can also described by percent sequence identity overthe full aligned length of the two polynucleotides which includespercent sequence identity of about at least 50%, 55%, 60%, 65%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100%. Sufficient homology includes any combination ofpolynucleotide length, global percent sequence identity, and optionallyconserved regions of contiguous nucleotides or local percent sequenceidentity, for example sufficient homology can be described as a regionof 75-150 bp having at least 80% sequence identity to a region of thetarget locus. Sufficient homology can also be described by the predictedability of two polynucleotides to specifically hybridize under highstringency conditions, see, for example, Sambrook et al., (1989)Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, NY); Current Protocols in Molecular Biology, Ausubel et al., Eds(1994) Current Protocols, (Greene Publishing Associates, Inc. and JohnWiley & Sons, Inc); and, Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, (Elsevier, N.Y.).

Any means can be used to bring together the various components needed toalter the genome of a dicot plant cell. For example, in in vitrosystems, the double-strand-break-inducing agent and thepolynucleotide(s) comprising the recognition site(s) can be provided bycontacting the components under the appropriate conditions for DNAcleavage.

Alternatively a variety of methods are known for the introduction ofnucleotide sequences and polypeptides into an organism, including, forexample, transformation, sexual crossing, and the introduction of thepolypeptide, DNA, or mRNA into the cell.

Methods for contacting, providing, and/or introducing a composition intovarious organisms are known and include but are not limited to, stabletransformation methods, transient transformation methods, virus-mediatedmethods, and sexual breeding. Stable transformation indicates that theintroduced polynucleotide integrates into the genome of the organism andis capable of being inherited by progeny thereof. Transienttransformation indicates that the introduced composition is onlytemporarily expressed or present in the organism.

Protocols for introducing polynucleotides and polypeptides into plantsmay vary depending on the type of plant or plant cell targeted fortransformation, such as monocot or dicot. Suitable methods ofintroducing polynucleotides and polypeptides into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al., (1986) Biotechniques 4:320-34 and U.S. Pat. No.6,300,543), meristem transformation (U.S. Pat. No. 5,736,369),electroporation (Riggs et al., (1986) Proc. Natl. Acad. Sci. USA83:5602-6, Agrobacterium-mediated transformation (U.S. Pat. Nos.5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al.,(1984) EMBO J 3:2717-22), and ballistic particle acceleration (U.S. Pat.Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al., (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg & Phillips (Springer-Verlag, Berlin); McCabe etal., (1988) Biotechnology 6:923-6; Weissinger et al., (1988) Ann RevGenet 22:421-77; Sanford et al., (1987) Particulate Science andTechnology 5:27-37 (onion); Christou et al., (1988) Plant Physiol87:671-4 (soybean); Finer and McMullen, (1991) In Vitro Cell Dev Biol27P:175-82 (soybean); Singh et al., (1998) Theor Appl Genet 96:319-24(soybean); Datta et al., (1990) Biotechnology 8:736-40 (rice); Klein etal., (1988) Proc. Natl. Acad. Sci. USA 85:4305-9 (maize); Klein et al.,(1988) Biotechnology 6:559-63 (maize); U.S. Pat. Nos. 5,240,855;5,322,783 and 5,324,646; Klein et al., (1988) Plant Physiol 91:440-4(maize); Fromm et al., (1990) Biotechnology 8:833-9 (maize);Hooykaas-Van Slogteren et al., (1984) Nature 311:763-4; U.S. Pat. No.5,736,369 (cereals); Bytebier et al., (1987) Proc. Natl. Acad. Sci. USA84:5345-9 (Liliaceae); De Wet et al., (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al., (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al., (1990) Plant Cell Rep 9:415-8) andKaeppler et al., (1992) Theor Appl Genet 84:560-6 (whisker-mediatedtransformation); D'Halluin et al., (1992) Plant Cell 4:1495-505(electroporation); Li et al., (1993) Plant Cell Rep 12:250-5; Christouand Ford (1995) Annals Botany 75:407-13 (rice) and Osjoda et al., (1996)Nat Biotechnol 14:745-50 (maize via Agrobacterium tumefaciens).

Alternatively, polynucleotides may be introduced into plants bycontacting plants with a virus or viral nucleic acids. Generally, suchmethods involve incorporating a polynucleotide within a viral DNA or RNAmolecule. In some examples a polypeptide of interest may be initiallysynthesized as part of a viral polyprotein, which is later processed byproteolysis in vivo or in vitro to produce the desired recombinantprotein. Methods for introducing polynucleotides into plants andexpressing a protein encoded therein, involving viral DNA or RNAmolecules, are known, see, for example, U.S. Pat. Nos. 5,889,191,5,889,190, 5,866,785, 5,589,367 and 5,316,931. Transient transformationmethods include, but are not limited to, the introduction ofpolypeptides, such as a double-strand break inducing agent, directlyinto the organism, the introduction of polynucleotides such as DNAand/or RNA polynucleotides, and the introduction of the RNA transcript,such as an mRNA encoding a double-strand break inducing agent, into theorganism. Such methods include, for example, microinjection or particlebombardment. See, for example Crossway et al., (1986) Mol Gen Genet202:179-85; Nomura et al., (1986) Plant Sci 44:53-8; Hepler et al.,(1994) Proc. Natl. Acad. Sci. USA 91:2176-80; and, Hush et al., (1994) JCell Sci 107:775-84.

Standard DNA isolation, purification, molecular cloning, vectorconstruction, and verification/characterization methods are wellestablished, see, for example Sambrook et al., (1989) Molecular Cloning:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY). Vectorsand constructs include circular plasmids, and linear polynucleotides,comprising a polynucleotide of interest and optionally other componentsincluding linkers, adapters, regulatory regions, introns, restrictionsites, enhancers, insulators, selectable markers, nucleotide sequencesof interest, promoters, and/or other sites that aid in vectorconstruction or analysis. In some examples a recognition site and/ortarget site can be contained within an intron, coding sequence, 5′ UTRs,3′ UTRs, and/or regulatory regions.

The present invention further provides expression constructs forexpressing in a plant, plant cell, or plant part an endonuclease that iscapable of binding to and creating a double strand break in a targetsite. The expression constructs of the invention comprise a promoteroperably linked to a nucleotide sequence encoding an endonuclease of thepresent invention. The promoter is capable of driving expression of anoperably linked nucleotide sequence in a plant cell. Any such promoterthat is disclosed herein or known in the art can be used in the presentinvention. In one embodiment, the target site of the endonuclease isselected from the group consisting of TS21, TS14, TS30, TSS, TS7, TS4,TS22, and TS24 target sites of soybean, which have the nucleotidesequences set forth in SEQ ID NO:1, 2, 3, 4, 5, 6 , 7, and 8,respectively. In another embodiment, the target site of the endonucleaseis selected from the group consisting of MHP1, MHP14, MHP32, MHP42,MHP55, MHP67, MHP77, MHP98, MHP107, and MHP115 target sites of maize,which have the nucleotide sequences set forth in SEQ ID NO:68, 69, 70,71, 72, 73, 74, 75, 76, and 77, respectively.

In certain embodiments, the expression constructs comprise a nucleotidesequence encoding the endonuclease that has been custom designed orengineered to cut at one the soybean target sites set forth above. Suchnucleotide sequences include, for example, the nucleotide sequences setforth in SEQ ID NOS:9, 10, 11, 12, 13, 14, 15, and 16. Other nucleotidesequences of the invention include, but are not limited to, nucleotidesequences comprising a coding sequence of a DNA binding domain of anendonuclease, wherein the coding sequence is nucleotides 100-261 andnucleotides 850-1011 of SEQ ID NO:9, 10, 11, 12, 13, 14, 15 or 16 anddegenerate coding sequences thereof. Such a degenerate coding sequenceencodes the same amino acid sequence as that encoded by one of thecoding sequences set forth in nucleotides 100-261 and nucleotides850-1011 of SEQ ID NO:9, 10, 11, 12, 13, 14, 15 or 16 but differs in itsnucleotide sequence due to the degeneracy of the genetic code.

In certain other embodiments, the expression constructs comprise anucleotide sequence encoding the endonuclease that has been customdesigned or engineered to cut at one the maize target sites set forthabove. Such nucleotide sequences include, for example, the nucleotidesequences set forth in SEQ ID NOS: 78, 79, 80, 81, 82, and 83. Othernucleotide sequences of the invention include, but are not limited to,nucleotide sequences comprising a coding sequence of a DNA bindingdomain of an endonuclease, wherein the coding sequence comprisesnucleotides 100-261 and nucleotides 850-1011 of SEQ ID NO: 80 anddegenerate coding sequences thereof. Such a degenerate coding sequenceencodes the same amino acid sequence as that encoded by one of thecoding sequences set forth in nucleotides 100-261 and nucleotides850-1011 of SEQ ID NO: 80 but differs in its nucleotide sequence due tothe degeneracy of the genetic code. Other nucleotide sequences of theinvention include, but are not limited to, nucleotide sequencescomprising a coding sequence of a DNA binding domain of an endonuclease,wherein the coding sequence is nucleotides 100-261 and nucleotides661-822 of SEQ ID NO: 78, 79, 81, 82 or 83 and degenerate codingsequences thereof. Such a degenerate coding sequence encodes the sameamino acid sequence as that encoded by one of the coding sequences setforth in nucleotides 100-261 and nucleotides 661-822 of SEQ ID NO: 78,79, 81, 82 or 83 but differs in its nucleotide sequence due to thedegeneracy of the genetic code.

Any promoter can be used, and can be selected based on the desiredoutcome. A promoter is a region of DNA involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A plant promoter is a promoter capable of initiating transcription in aplant cell, for a review of plant promoters, see, Potenza et al., (2004)In Vitro Cell Dev Biol 40:1-22. Constitutive promoters include, forexample, the core promoter of the Rsyn7 promoter and other constitutivepromoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the coreCaMV 35S promoter (Odell et al., (1985) Nature 313:810-2); rice actin(McElroy et al., (1990) Plant Cell 2:163-71); ubiquitin (Christensen etal., (1989) Plant Mol Biol 12:619-32; Christensen et al., (1992) PlantMol Biol 18:675-89); pEMU (Last et al., (1991) Theor Appl Genet81:581-8); MAS (Velten et al., (1984) EMBO J 3:2723-30); ALS promoter(U.S. Pat. No. 5,659,026), and the like. Other constitutive promotersare described in, for example, U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and6,177,611. In some examples an inducible promoter may be used.Pathogen-inducible promoters induced following infection by a pathogeninclude, but are not limited to those regulating expression of PRproteins, SAR proteins, beta-1,3-glucanase, chitinase, etc.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. The promoter may be a chemical-inducible promoter, whereapplication of the chemical induces gene expression, or achemical-repressible promoter, where application of the chemicalrepresses gene expression. Chemical-inducible promoters include, but arenot limited to, the maize In2-2 promoter, activated by benzenesulfonamide herbicide safeners (De Veylder et al., (1997) Plant CellPhysiol 38:568-77), the maize GST promoter (GST-II-27, WO93/01294),activated by hydrophobic electrophilic compounds used as pre-emergentherbicides, and the tobacco PR-1a promoter (Ono et al., (2004) BiosciBiotechnol Biochem 68:803-7) activated by salicylic acid. Otherchemical-regulated promoters include steroid-responsive promoters (see,for example, the glucocorticoid-inducible promoter (Schena et al.,(1991) Proc. Natl. Acad. Sci. USA 88:10421-5; McNellis et al., (1998)Plant J 14:247-257); tetracycline-inducible and tetracycline-repressiblepromoters (Gatz et al., (1991) Mol Gen Genet 227:229-37; U.S. Pat. Nos.5,814,618 and 5,789,156).

Tissue-preferred promoters can be utilized to target enhanced expressionwithin a particular plant tissue. Tissue-preferred promoters include,for example, Kawamata et al., (1997) Plant Cell Physiol 38:792-803;Hansen et al., (1997) Mol Gen Genet 254:337-43; Russell et al., (1997)Transgenic Res 6:157-68; Rinehart et al., (1996) Plant Physiol112:1331-41; Van Camp et al., (1996) Plant Physiol 112:525-35;Canevascini et al., (1996) Plant Physiol 112:513-524; Lam, (1994)Results Probl Cell Differ 20:181-96; and Guevara-Garcia et al., (1993)Plant J 4:495-505. Leaf-preferred promoters include, for example,Yamamoto et al., (1997) Plant J 12:255-65; Kwon et al., (1994) PlantPhysiol 105:357-67; Yamamoto et al., (1994) Plant Cell Physiol 35:773-8;Gotor et al., (1993) Plant J 3:509-18; Orozco et al., (1993) Plant MolBiol 23:1129-38; Matsuoka et al., (1993) Proc. Natl. Acad. Sci. USA90:9586-90; Simpson et al., (1958) EMBO J 4:2723-9; Timko et al., (1988)Nature 318:57-8. Root-preferred promoters include, for example, Hire etal., (1992) Plant Mol Biol 20:207-18 (soybean root-specific glutaminesynthase gene); Miao et al., (1991) Plant Cell 3:11-22 (cytosolicglutamine synthase (GS)); Keller and Baumgartner, (1991) Plant Cell3:1051-61 (root-specific control element in the GRP 1.8 gene of Frenchbean); Sanger et al., (1990) Plant Mol Biol 14:433-43 (root-specificpromoter of A. tumefaciens mannopine synthase (MAS)); Bogusz et al.,(1990) Plant Cell 2:633-41 (root-specific promoters isolated fromParasponia andersonii and Trema tomentosa); Leach and Aoyagi, (1991)Plant Sci 79:69-76 (A. rhizogenes roIC and roID root-inducing genes);Teeri et al., (1989) EMBO J 8:343-50 (Agrobacterium wound-induced TR1′and TR2′ genes); VfENOD-GRP3 gene promoter (Kuster et al., (1995) PlantMol Biol 29:759-72); and roIB promoter (Capana et al., (1994) Plant MolBiol 25:681-91; phaseolin gene (Murai et al., (1983) Science 23:476-82;Sengopta-Gopalen et al., (1988) Proc. Natl. Acad. Sci. USA 82:3320-4).See also, U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252;5,401,836; 5,110,732 and 5,023,179.

Seed-preferred promoters include both seed-specific promoters activeduring seed development, as well as seed-germinating promoters activeduring seed germination. See, Thompson et al., (1989) BioEssays 10:108.Seed-preferred promoters include, but are not limited to, Cim1(cytokinin-induced message); cZ19B1 (maize 19 kDa zein); and milps(myo-inositol-1-phosphate synthase); (WO00/11177; and U.S. Pat. No.6,225,529). For dicots, seed-preferred promoters include, but are notlimited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin,cruciferin, and the like. For monocots, seed-preferred promotersinclude, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDagamma zein, waxy, shrunken 1, shrunken 2, globulin 1, oleosin, and nuc1.See also, WO00/12733, where seed-preferred promoters from END1 and END2genes are disclosed.

A phenotypic marker is screenable or selectable marker that includesvisual markers and selectable markers whether it is a positive ornegative selectable marker. Any phenotypic marker can be used.Specifically, a selectable or screenable marker comprises a DNA segmentthat allows one to identify, or select for or against a molecule or acell that contains it, often under particular conditions. These markerscan encode an activity, such as, but not limited to, production of RNA,peptide, or protein, or can provide a binding site for RNA, peptides,proteins, inorganic and organic compounds or compositions and the like.

Examples of selectable markers include, but are not limited to, DNAsegments that comprise restriction enzyme sites; DNA segments thatencode products which provide resistance against otherwise toxiccompounds including antibiotics, such as, spectinomycin, ampicillin,kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO) andhygromycin phosphotransferase (HPT)); DNA segments that encode productswhich are otherwise lacking in the recipient cell (e.g., tRNA genes,auxotrophic markers); DNA segments that encode products which can bereadily identified (e.g., phenotypic markers such as β-galactosidase,GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan(CFP), yellow (YFP), red (RFP), and cell surface proteins); thegeneration of new primer sites for PCR (e.g., the juxtaposition of twoDNA sequence not previously juxtaposed), the inclusion of DNA sequencesnot acted upon or acted upon by a restriction endonuclease or other DNAmodifying enzyme, chemical, etc.; and, the inclusion of a DNA sequencesrequired for a specific modification (e.g., methylation) that allows itsidentification.

Additional selectable markers include genes that confer resistance toherbicidal compounds, such as glufosinate ammonium, bromoxynil,imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See for example,Yarranton, (1992) Curr Opin Biotech 3:506-11; Christopherson et al.,(1992) Proc. Natl. Acad. Sci. USA 89:6314-8; Yao et al., (1992) Cell71:63-72; Reznikoff, (1992) Mol Microbiol 6:2419-22; Hu et al., (1987)Cell 48:555-66; Brown et al., (1987) Cell 49:603-12; Figge et al.,(1988) Cell 52:713-22; Deuschle et al., (1989) Proc. Natl. Acad. Sci.USA 86:5400-4; Fuerst et al., (1989) Proc. Natl. Acad. Sci. USA86:2549-53; Deuschle et al., (1990) Science 248:480-3; Gossen, (1993)Ph.D. Thesis, University of Heidelberg; Reines et al., (1993) Proc.Natl. Acad. Sci. USA 90:1917-21; Labow et al., (1990) Mol Cell Biol10:3343-56; Zambretti et al., (1992) Proc. Natl. Acad. Sci. USA89:3952-6; Baim et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-6;Wyborski et al., (1991) Nucleic Acids Res 19:4647-53; Hillen andWissman, (1989) Topics Mol Struc Biol 10:143-62; Degenkolb et al.,(1991) Antimicrob Agents Chemother 35:1591-5; Kleinschnidt et al.,(1988) Biochemistry 27:1094-104; Bonin, (1993) Ph.D. Thesis, Universityof Heidelberg; Gossen et al., (1992) Proc. Natl. Acad. Sci. USA89:5547-51; Oliva et al., (1992) Antimicrob Agents Chemother 36:913-9;Hlavka et al., (1985) Handbook of Experimental Pharmacology, Vol. 78(Springer-Verlag, Berlin); Gill et al., (1988) Nature 334:721-4.

The cells having the introduced sequence may be grown or regeneratedinto plants using conventional conditions, see for example, McCormick etal., (1986) Plant Cell Rep 5:81-4. These plants may then be grown, andeither pollinated with the same transformed strain or with a differenttransformed or untransformed strain, and the resulting progeny havingthe desired characteristic and/or comprising the introducedpolynucleotide or polypeptide identified. Two or more generations may begrown to ensure that the polynucleotide is stably maintained andinherited, and seeds harvested.

Any plant can be used, including moncot and dicot plants. Examples ofmonocot plants that can be used include, but are not limited to, corn(Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), wheat (Triticum aestivum),sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), switchgrass(Panicum virgatum), pineapple (Ananas comosus), banana (Musa spp.),palm, ornamentals, turfgrasses, and other grasses. Examples of dicotplants that can be used include, but are not limited to, soybean(Glycine max), canola (Brassica napus and B. campestris), alfalfa(Medicago sativa), tobacco (Nicotiana tabacum), Arabidopsis (Arabidopsisthaliana), sunflower (Helianthus annuus), cotton (Gossypium arboreum),and peanut (Arachis hypogaea), tomato (Solanum lycopersicum), potato(Solanum tuberosum) etc.

The transgenes, recombinant DNA molecules, DNA sequences of interest,and polynucleotides of interest can comprise one or more genes ofinterest. Such genes of interest can encode, for example, a protein thatprovides agronomic advantage to the plant. Genes of interest, including,but not limited to, those that encode proteins that provide agronomicadvantage, can be reflective of the commercial markets and interests ofthose involved in the development of the crop. Crops and markets ofinterest change, and as developing nations open up world markets, newcrops and technologies will emerge also. In addition, as ourunderstanding of agronomic traits and characteristics such as yield andheterosis increase, the choice of genes for transformation will changeaccordingly. General categories of genes of interest include, forexample, those genes involved in information, such as zinc fingers,those involved in communication, such as kinases, and those involved inhousekeeping, such as heat shock proteins. More specific categories oftransgenes, for example, include genes encoding important traits foragronomics, insect resistance, disease resistance, herbicide resistance,sterility, grain characteristics, and commercial products. Genes ofinterest include, generally, those involved in oil, starch,carbohydrate, or nutrient metabolism as well as those affecting kernelsize, sucrose loading, and the like.

Agronomically important traits such as oil, starch, and protein contentcan be genetically altered in addition to using traditional breedingmethods. Modifications include increasing content of oleic acid,saturated and unsaturated oils, increasing levels of lysine and sulfur,providing essential amino acids, and also modification of starch.Hordothionin protein modifications are described in U.S. Pat. Nos.5,703,049, 5,885,801, 5,885,802, and 5,990,389, herein incorporated byreference. Another example is lysine and/or sulfur rich seed proteinencoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016,and the chymotrypsin inhibitor from barley, described in Williamson etal. (1987) Eur. J. Biochem. 165:99-106, the disclosures of which areherein incorporated by reference.

Derivatives of the coding sequences can be made by site-directedmutagenesis to increase the level of preselected amino acids in theencoded polypeptide. For example, the gene encoding the barley highlysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor,U.S. application Ser. No. 08/740,682, filed Nov. 1, 1996, and WO98/20133, the disclosures of which are herein incorporated by reference.Other proteins include methionine-rich plant proteins such as fromsunflower seed (Lilley et al. (1989) Proceedings of the World Congresson Vegetable Protein Utilization in Human Foods and Animal Feedstuffs,ed. Applewhite (American Oil Chemists Society, Champaign, Illinois), pp.497-502; herein incorporated by reference); corn (Pedersen et al. (1986)J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; both ofwhich are herein incorporated by reference); and rice (Musumura et al.(1989) Plant Mol. Biol. 12:123, herein incorporated by reference). Otheragronomically important genes encode latex, Floury 2, growth factors,seed storage factors, and transcription factors.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer, and the like.Such genes include, for example, Bacillus thuringiensis toxic proteingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;5,593,881; and Geiser et al. (1986) Gene 48:109); and the like.

Genes encoding disease resistance traits include detoxification genes,such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr)and disease resistance (R) genes (Jones et al. (1994) Science 266:789;Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell78:1089); and the like.

Herbicide resistance traits may include genes coding for resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides (e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance, in particular the S4 and/or Hra mutations), genes coding forresistance to herbicides that act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene);glyphosate (e.g., the EPSPS gene and the GAT gene; see, for example,U.S. Publication No. 20040082770 and WO 03/092360); or other such genesknown in the art. The bar gene encodes resistance to the herbicidebasta, the nptII gene encodes resistance to the antibiotics kanamycinand geneticin, and the ALS-gene mutants encode resistance to theherbicide chlorsulfuron.

Sterility genes can also be encoded in an expression cassette andprovide an alternative to physical detasseling. Examples of genes usedin such ways include male tissue-preferred genes and genes with malesterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210.Other genes include kinases and those encoding compounds toxic to eithermale or female gametophytic development.

The quality of grain is reflected in traits such as levels and types ofoils, saturated and unsaturated, quality and quantity of essential aminoacids, and levels of cellulose. In corn, modified hordothionin proteinsare described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and5,990,389.

Commercial traits can also be encoded on a gene or genes that couldincrease for example, starch for ethanol production, or provideexpression of proteins. Another important commercial use of transformedplants is the production of polymers and bioplastics such as describedin U.S. Pat. No. 5,602,321. Genes such as β-Ketothiolase, PHBase(polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase (seeSchubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitateexpression of polyhyroxyalkanoates (PHAs).

Exogenous products include plant enzymes and products as well as thosefrom other sources including procaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like. The levelof proteins, particularly modified proteins having improved amino aciddistribution to improve the nutrient value of the plant, can beincreased. This is achieved by the expression of such proteins havingenhanced amino acid content.

The transgenes, recombinant DNA molecules, DNA sequences of interest,and polynucleotides of interest can be comprise one or more DNAsequences for gene silencing. Methods for gene silencing involving theexpression of DNA sequences in plant are known in the art include, butare not limited to, cosuppression, antisense suppression,double-stranded RNA (dsRNA) interference, hairpin RNA (hpRNA)interference, intron-containing hairpin RNA (ihpRNA) interference,transcriptional gene silencing, and micro RNA (miRNA) interference

Cosuppression may be used to inhibit the expression of plant genes toproduce plants having undetectable protein levels for the proteinsencoded by these genes. See, for example, Broin et al. (2002) Plant Cell14:1417-1432. Cosuppression may also be used to inhibit the expressionof multiple proteins in the same plant. See, for example, U.S. Pat. No.5,942,657. Methods for using cosuppression to inhibit the expression ofendogenous genes in plants are described in Flavell et al. (1994) Proc.Natl. Acad. Sci. USA 91:3490-3496; Jorgensen et al. (1996) Plant Mol.Biol. 31:957-973; Johansen and Carrington (2001) Plant Physiol.126:930-938; Broin et al. (2002) Plant Cell 14:1417-1432; Stoutjesdijket al (2002) Plant Physiol. 129:1723-1731; Yu et al. (2003)Phytochemistry 63:753-763; and U.S. Pat. Nos. 5,034,323, 5,283,184, and5,942,657; each of which is herein incorporated by reference. Theefficiency of cosuppression may be increased by including a poly-dTregion in the expression cassette at a position 3′ to the sense sequenceand 5′ of the polyadenylation signal. See, U.S. Patent Publication No.20020048814, herein incorporated by reference. Typically, such anucleotide sequence has substantial sequence identity to the sequence ofthe transcript of the endogenous gene, optimally greater than about 65%sequence identity, more optimally greater than about 85% sequenceidentity, most optimally greater than about 95% sequence identity. See,U.S. Pat. Nos. 5,283,184 and 5,034,323; herein incorporated byreference.

Antisense suppression may be used to inhibit the expression of multipleproteins in the same plant. See, for example, U.S. Pat. No. 5,942,657.Furthermore, portions of the antisense nucleotides may be used todisrupt the expression of the target gene. Generally, sequences of atleast 50 nucleotides, 100 nucleotides, 200 nucleotides, 300, 400, 450,500, 550, or greater may be used. Methods for using antisensesuppression to inhibit the expression of endogenous genes in plants aredescribed, for example, in Liu et al (2002) Plant Physiol. 129:1732-1743and U.S. Pat. Nos. 5,759,829 and 5,942,657, each of which is hereinincorporated by reference. Efficiency of antisense suppression may beincreased by including a poly-dT region in the expression cassette at aposition 3′ to the antisense sequence and 5′ of the polyadenylationsignal. See, U.S. Patent Publication No. 20020048814, hereinincorporated by reference.

Methods for using dsRNA interference to inhibit the expression ofendogenous plant genes are described in Waterhouse et al. (1998) Proc.Natl. Acad. Sci. USA 95:13959-13964, Liu et al. (2002) Plant Physiol.129:1732-1743, and WO 99/49029, WO 99/53050, WO 99/61631, and WO00/49035; each of which is herein incorporated by reference.

Methods of hpRNA interference are described in Waterhouse and Helliwell(2003) Nat. Rev. Genet. 4:29-38 and the references cited therein. Thesemethods are highly efficient at inhibiting the expression of endogenousgenes. See, for example, Chuang and Meyerowitz (2000) Proc. Natl. Acad.Sci. USA 97:4985-4990; Stoutjesdijk et al. (2002) Plant Physiol.129:1723-1731; and Waterhouse and Helliwell (2003) Nat. Rev. Genet.4:29-38. Methods for using hpRNA interference to inhibit or silence theexpression of genes are described, for example, in Chuang and Meyerowitz(2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijk et al.(2002) Plant Physiol. 129:1723-1731; Waterhouse and Helliwell (2003)Nat. Rev. Genet. 4:29-38; Pandolfini et al. BMC Biotechnology 3:7, andU.S. Patent Publication No. 20030175965; each of which is hereinincorporated by reference. A transient assay for the efficiency of hpRNAconstructs to silence gene expression in vivo has been described byPanstruga et al. (2003) Mol. Biol. Rep. 30:135-140, herein incorporatedby reference.

For ihpRNA, the interfering molecules have the same general structure asfor hpRNA, but the RNA molecule additionally comprises an intron that iscapable of being spliced in the cell in which the ihpRNA is expressed.The use of an intron minimizes the size of the loop in the hairpin RNAmolecule following splicing, and this increases the efficiency ofinterference. See, for example, Smith et al. (2000) Nature 407:319-320.In fact, Smith et al. show 100% suppression of endogenous geneexpression using ihpRNA-mediated interference. Methods for using ihpRNAinterference to inhibit the expression of endogenous plant genes aredescribed, for example, in Smith et al. (2000) Nature 407:319-320;Wesley et al. (2001) Plant J. 27:581-590; Wang and Waterhouse (2001)Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell (2003) Nat.Rev. Genet. 4:29-38; Helliwell and Waterhouse (2003) Methods 30:289-295,and U.S. Patent Publication No. 20030180945, each of which is hereinincorporated by reference.

Transcriptional gene silencing (TGS) may be accomplished through use ofhpRNA constructs wherein the inverted repeat of the hairpin sharessequence identity with the promoter region of a gene to be silenced.Processing of the hpRNA into short RNAs which can interact with thehomologous promoter region may trigger degradation or methylation toresult in silencing (Aufsatz et al. (2002) PNAS 99 (Suppl.4):16499-16506; Mette et al. (2000) EMBO J 19(19):5194-5201).

The inhibition of the expression of a target protein may be obtained byRNA interference by expression of a gene encoding a micro RNA (miRNA).miRNAs are regulatory agents consisting of about 22 ribonucleotides.miRNA are highly efficient at inhibiting the expression of endogenousgenes. See, for example Javier et al. (2003) Nature 425: 257-263, hereinincorporated by reference. For miRNA interference, the expressioncassette is designed to express an RNA molecule that is modeled on anendogenous miRNA gene. The miRNA gene encodes an RNA that forms ahairpin structure containing a 22-nucleotide sequence that iscomplementary to another endogenous gene (target sequence). miRNAmolecules are highly efficient at inhibiting the expression ofendogenous genes, and the RNA interference they induce is inherited bysubsequent generations of plants.

The frequency of homologous recombination is influenced by a number offactors. Different organisms vary with respect to the amount ofhomologous recombination and the relative proportion of homologous tonon-homologous recombination. Generally, the length of the region ofhomology affects the frequency of homologous recombination events, thelonger the region of homology, the greater the frequency. The length ofthe homology region needed to observe homologous recombination is alsospecies-variable. In many cases, at least 5 kb of homology has beenutilized, but homologous recombination has been observed with as littleas 25-50 bp of homology. The minimum length of homology needed has beenestimated at 20-50 bp in E. coli (Singer et al., (1982) Cell 31:25-33;Shen and Huang, (1986) Genetics 112:441-57; Watt et al., (1985) Proc.Natl. Acad. Sci. USA 82:4768-72), 63-89 bp in Sacchromyces. cerevisaie(Sugawara and Haber, (1992) Mol Cell Biol 12:563-75), and 163-300 bp inmammalian cells (Rubnitz and Subramani, (1984) Mol Cell Biol 4:2253-8;Ayares et al., (1986) Proc. Natl. Acad. Sci. USA 83:5199-203; Liskay etal., (1987) Genetics 115:161-7).

Homologous recombination has been demonstrated in insects. InDrosophila, Dray and Gloor found that as little as 3 kb of totaltemplate:target homology sufficed to copy a large non-homologous segmentof DNA into the target with reasonable efficiency (Dray and Gloor,(1997) Genetics 147:689-99). Using FLP-mediated DNA integration at atarget FRT in Drosophila, Golic et al., showed integration wasapproximately 10-fold more efficient when the donor and target shared4.1 kb of homology as compared to 1.1 kb of homology (Golic et al.,(1997) Nucleic Acids Res 25:3665). Data from Drosophila indicates that2-4 kb of homology is sufficient for efficient targeting, but there issome evidence that much less homology may suffice, on the order of about30 bp to about 100 bp (Nassif and Engels, (1993) Proc. Natl. Acad. Sci.USA 90:1262-6; Keeler and Gloor, (1997) Mol Cell Biol 17:627-34).

Homologous recombination has also been accomplished in other organisms.For example, at least 150-200 bp of homology was required for homologousrecombination in the parasitic protozoan Leishmania (Papadopoulou andDumas, (1997) Nucleic Acids Res 25:4278-86). In the filamentous fungusAspergillus nidulans, gene replacement has been accomplished with aslittle as 50 bp flanking homology (Chaveroche et al., (2000) NucleicAcids Res 28:e97). Targeted gene replacement has also been demonstratedin the ciliate Tetrahymena thermophila (Gaertig et al., (1994) NucleicAcids Res 22:5391-8). In mammals, homologous recombination has been mostsuccessful in the mouse using pluripotent embryonic stem cell lines (ES)that can be grown in culture, transformed, selected and introduced intoa mouse embryo. Embryos bearing inserted transgenic ES cells develop asgenetically chimeric offspring. By interbreeding siblings, homozygousmice carrying the selected genes can be obtained. An overview of theprocess is provided in Watson et al., (1992) Recombinant DNA, 2nd Ed.,(Scientific American Books distributed by WH Freeman & Co.); Capecchi,(1989) Trends Genet 5:70-6; and Bronson, (1994) J Biol Chem 269:27155-8.Homologous recombination in mammals other than mouse has been limited bythe lack of stem cells capable of being transplanted to oocytes ordeveloping embryos. However, McCreath et al., Nature 405:1066-9 (2000)reported successful homologous recombination in sheep by transformationand selection in primary embryo fibroblast cells.

Error-prone DNA repair mechanisms can produce mutations at double-strandbreak sites. The nonhomologous end-joining (NHEJ) pathways are the mostcommon repair mechanism to bring the broken ends together (Bleuyard etal., (2006) DNA Repair 5:1-12). The structural integrity of chromosomesis typically preserved by the repair, but deletions, insertions, orother rearrangements are possible. The two ends of one double-strandbreak are the most prevalent substrates of NHEJ (Kirik et al., (2000)EMBO J 19:5562-6), however if two different double-strand breaks occur,the free ends from different breaks can be ligated and result inchromosomal deletions (Siebert and Puchta, (2002) Plant Cell14:1121-31), or chromosomal translocations between different chromosomes(Pacher et al., (2007) Genetics 175:21-9).

Episomal DNA molecules can also be ligated into the double-strand break,for example, integration of T-DNAs into chromosomal double-strand breaks(Chilton and Que, (2003) Plant Physiol 133:956-65; Salomon and Puchta,(1998) EMBO J 17:6086-95). Once the sequence around the double-strandbreaks is altered, for example, by exonuclease activities involved inthe maturation of double-strand breaks, gene conversion pathways canrestore the original structure if a homologous sequence is available,such as a homologous chromosome in non-dividing somatic cells, or asister chromatid after DNA replication (Molinier et al., (2004) PlantCell 16:342-52). Ectopic and/or epigenic DNA sequences may also serve asa DNA repair template for homologous recombination (Puchta, (1999)Genetics 152:1173-81).

Alteration of the genome of a plant cell, for example, throughhomologous recombination (HR), is a powerful tool for geneticengineering. Despite the low frequency of homologous recombination inhigher plants, there are a few examples of successful homologousrecombination of plant endogenous genes. The parameters for homologousrecombination in plants have primarily been investigated by rescuingintroduced truncated selectable marker genes. In these experiments, thehomologous DNA fragments were typically between 0.3 kb to 2 kb. Observedfrequencies for homologous recombination were on the order of 10⁻⁴ to10⁻⁵. See, for example, Halfter et al., (1992) Mol Gen Genet 231:186-93;Offringa et al., (1990) EMBO J 9:3077-84; Offringa et al., (1993) Proc.Natl. Acad. Sci. USA 90:7346-50; Paszkowski et al., (1988) EMBO J7:4021-6; Hourda and Paszkowski, (1994) Mol Gen Genet 243:106-11; andRisseeuw et al., (1995) Plant J 7:109-19.

An endogenous, non-selectable gene was targeted in Arabidopsis using atargeting vector containing a region of about 7 kb homologous to thetarget gene and the targeting frequency was estimated to be at least3.9×10⁻⁴ (Maio and Lam, (1995) Plant J 7:359-65). In another example,using a positive-negative selection scheme and a targeting vectorcontaining up to 22.9 kb of sequence homologous to the target,homologous recombination was detected with a frequency less than5.3×10⁻⁵, despite the large flanking sequences available forrecombination (Thykær et al., (1997) Plant Mol Biol 35:523-30). InArabidopsis, the AGL5 MADS-box gene was knocked out by homologousrecombination using a targeting construct consisting of akanamycin-resistance cassette inserted into the AGL5 sequence roughly 3kb from the 5′ end and 2 kb from the 3′ end. Of the 750kanamycin-resistant transgenic lines that were generated, one linecontained the anticipated insertion (Kempin et al., (1997) Nature389:802-3). Hanin et al., obtained homologous recombination events at abasal frequency of 7×10⁻⁴ using 3 kb 5′-end and 2 kb 3′-end homology tothe Arabidopsis PPO gene encoding protoporphyrinogen oxidase (Hanin etal., (2001) Plant J 28:671-7). Terada et al., targeted the Waxy locus inrice using an Agrobacterium-mediated transformation procedure. Negativeselection, in the form of two copies of the diphteria toxin gene placedat both ends of T-DNA, was used to eliminate random integration ofT-DNAs, allowing for enrichment of rare homologous recombination eventsin the selected material, and their transformation system generatedthousands of events from just 150 rice seeds. The reported frequency ofhomologous recombination of the waxy gene in rice was 0.65×10⁻³, withoutinclusion of elements to enhance homologous recombination (Terada etal., (2002) Nat Biotech 20:1030-4).

DNA double-strand breaks (DSBs) appear to be an effective factor tostimulate homologous recombination pathways in every organism tested todate (Puchta et al., (1995) Plant Mol Biol 28:281-92; Tzfira and White,(2005) Trends Biotechnol 23:567-9; Puchta, (2005) J Exp Bot 56:1-14).Using DNA-breaking agents, two- to nine-fold increase of homologousrecombination was observed between artificially constructed homologousDNA repeats in plants (Puchta et al., (1995) Plant Mol Biol 28:281-92).In maize protoplasts, experiments with linear DNA molecules demonstratedenhanced homologous recombination between plasm ids (Lyznik et al.,(1991) Mol Gen Genet 230:209-18).

The effects of DSBs on homologous recombination have been investigatedby using rare-cutting enzymes as well as transposons such as Ac andMutator (Chiurazzi et al., (1996) Plant Cell 8:2057-66; Puchta et al.,(1996) Proc. Natl. Acad. Sci. USA 93:5055-60; Xiao and Peterson, (2000)Mol Gen Genet 263:22-9; and Shalev and Levy (1997) Genetics146:1143-51). Chiurazzi et al., (1996) Plant Cell 8:2057-66) introducedDSBs into an Arabidopsis chromosome using HO-endonuclease and observed10-fold increase in the frequency of homologous recombination betweenrepeats flanking the HO recognition site. Excision of Ac transposableelements also stimulated homologous recombination between repeatsflanking the elements at an even higher frequency (Xiao and Peterson(2000) Mol Gen Genet 263:22-9).

Puchta et al. reported that homologous recombination frequency at anartificial target locus was increased by up to two orders of magnitudewhen DSBs were generated using I-SceI (Puchta et al., (1996) Proc. Natl.Acad. Sci. USA 93:5055-60). In the experiment reported in Puchta et al.,an I-SceI expression cassette was introduced into transgenic tobaccotarget lines together with targeting construct by co-inoculation withthe two respective Agrobacterium strains. Homologous recombinationbetween T-DNA containing the targeting construct and the target sitereconstituted the kanamycin-resistance gene (nptII). There was anapparent correlation between frequency of homologous recombination andthe amount of I-SceI expression cassette, suggesting that more DSBsyielded higher homologous recombination frequency.

High frequency of homologous recombination at a pre-introducedartificial target site was obtained using a zinc-finger nuclease (ZFN)in tobacco (Wright et al., (2005) Plant J 44:693-705). The zinc-fingernuclease expression cassette and donor DNA were introduced intoprotoplasts by co-electroporation and targeted modification wasmonitored by kanamycin resistance and GUS activity. One modified eventwas observed in approximately every 10 transformants, however, only 20%of the modified events contained the desired homologous recombinationproducts as indicated by Southern blot analysis.

Zinc finger nucleases are engineered endonucleases with alteredspecificities, for example by fusion of an engineered DNA binding domainto an endonuclease, for example, FokI (Durai et al., (2005) NucleicAcids Res 33:5978-90; Mani et al., (2005) Biochem Biophys Res Comm335:447-57). Wright et al., and Lloyd et al., reported a high frequencymutagenesis at a DNA target site integrated into tobacco or Arabidopsischromosomal DNA using zinc-finger nucleases (Wright et al., (2005) PlantJ 44:693-705; Lloyd et al., (2005) Proc. Natl. Acad. Sci. USA102:2232-7). Using a designed zinc-finger nuclease recognizing a tobaccoendogenous acetolactate synthase (ALS) gene locus, a mutated ALS geneknown to confer resistance to imidazolinone and sulphonylurea herbicideswas introduced to replace the endogenous ALS gene at frequenciesexceeding 2% of transformed cells (Townsend et al., (2009) Nature459:442-5). The knock-out of an endogenous gene and the expression of atransgene can be achieved simultaneously by gene targeting. The IPK1gene, which encodes inositol-1,3,4,5,6-pentakisphosphate 2-kinase neededin the final step of phytate biosythesis in maize seeds, was targetedusing a designed zinc-finger nuclease to insert via homologousrecombination a PAT gene, which encodes phosphinothricin acetyltransferase tolerance to glufosinate ammonium herbicides such asbialaphos. The disruption of the IPK1 gene with the insertion of the PATgene resulted in both herbicide tolerance and the expected alteration ofthe inositol phosphate profile in developing seeds (Shukla et al.,(2009) Nature 459:437-41).

Members of the serine family of recombinases produce double-strandbreaks at the recombination sites as a part of their catalyticactivities (Grindley et al., (2006) Ann Rev Biochem 16:16). The R/RSsystem in sweet orange appeared to induce mutations of RS sites leadingto chromosomal deletions not associated with site-specific recombinationreactions per se (Ballester et al., (2006) Plant Cell Rep 26:39-45).

Another approach uses protein engineering of existing homingendonucleases to alter their target specificities. Homing endonucleases,such as I-SceI or I-CreI, bind to and cleave relatively long DNArecognition sequences (18 bp and 22 bp, respectively). These sequencesare predicted to naturally occur infrequently in a genome, typicallyonly 1 or 2 sites/genome. The cleavage specificity of a homingendonuclease can be changed by rational design of amino acidsubstitutions at the DNA binding domain and/or combinatorial assemblyand selection of mutated monomers (see, for example, Arnould et al.,(2006) J Mol Biol 355:443-58; Ashworth et al., (2006) Nature 441:656-9;Doyon et al., (2006) J Am Chem Soc 128:2477-84; Rosen et al., (2006)Nucleic Acids Res 34:4791-800; and Smith et al., (2006) Nucleic AcidsRes 34:e149; Lyznik et al., (2009) U.S. Patent Application PublicationNo. 20090133152A1; Smith et al., (2007) U.S. Patent ApplicationPublication No. 20070117128A1). Engineered meganucleases have beendemonstrated that can cleave cognate mutant sites without broadeningtheir specificity. An artificial recognition site specific to the wildtype yeast I-SceI homing nuclease was introduced in maize genome andmutations of the recognition sequence were detected in 1% of analyzed F1plants when a transgenic I-SceI was introduced by crossing and activatedby gene excision (Yang et al., (2009) Plant Mol Biol 70:669-79). Morepractically, the maize liguleless locus was targeted using an engineeredsingle-chain endonuclease designed based on the I-CreI meganucleasesequence. Mutations of the selected liguleless locus recognitionsequence were detected in 3% of the T0 transgenic plants when thedesigned homing nuclease was introduced by Agrobacterium-mediatedtransformation of immature embryos (Gao et al., (2010) Plant J61:176-87).

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating embodiments of the invention, are given by way ofillustration only. From the above discussion and these Examples, oneskilled in the art can ascertain the essential characteristics of thisinvention, and without departing from the spirit and scope thereof, canmake various changes and modifications of the invention to adapt it tovarious usages and conditions. Such modifications are also intended tofall within the scope of the appended claims.

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s) and “kb” means kilobase(s).

The DNA repair mechanisms of cells are the basis of transformation tointroduce extraneous DNA or induce mutations on endogenous genes. DNAhomologous recombination is a specialized way of DNA repair that thecells repair

DNA damages using a homologous sequence. In plants, DNA homologousrecombination happens at frequencies too low to be used intransformation until it has been found that the process can bestimulated by DNA double-strand breaks (Bibikova et al., (2001) Mol.Cell Biol. 21:289-297; Puchta and Baltimore, (2003) Science 300:763;Wright et al., (2005) Plant J. 44:693-705).

Example 1 DNA Double-Strand-Break-Induced Alteration of an EndogenousTarget Site

When a DNA double-strand-break-inducing agent recognizes and cleaves thespecific recognition sequence at a target site in the genome, a DNAdouble-strand break is formed triggering the cell DNA repair mechanismsto mobilize to repair the damage that could be fatal to the cell. Theprocess can be utilized in plant transformation to introduce mutationsspecifically at the target site to knock out the gene residing at thetarget site or to insert a donor DNA of interest at the target site.Once the DNA double-strand break is formed, depending on the designs ofthe DNA constructs involved and the actual processes of DNA repair,different outcomes can be obtained serving different transformationpurposes.

For simple site-specific gene mutations, a target site containing arecognition sequence (FIG. 1A) and a DNA double-strand break agent suchas a endonuclease (FIG. 1B) that recognizes specifically the recognitionsequence have to be present in the same cell. After the endonucleaserecognizes and cuts the DNA, the two free ends can be repaired throughend joining by the cell DNA repair machinery without the intervention ofany external factors. The two ends can be repaired to its original stateso no change can be detected or they can be altered before beingrepaired resulting detectable changes after they are connected againsuch as the deletion of one or more nucleotides of the recognitionsequence and possibly extra surrounding sequences (FIG. 1F). Mutationsare introduced at the target site by the latter process.

To achieve site-specific DNA insertions, a donor DNA containing the DNAof interest has to be simultaneously present in the cell in addition tothe target site and the endonuclease. The donor DNA can contain the sameDNA sequences that flank the target site to flank the gene of interest,i.e., the homologous sequences (FIG. 1C). The DNA of interest can beinserted at the target site by homologous recombination (FIG. 1E), aprocess that is stimulated by the DNA double-strand break at the targetsite. The donor DNA can also contain only the DNA of interest withoutany flanking homologous sequences (FIG. 1D). The DNA of interest canstill be inserted at the target site though in a less predictablefashion through non-homologous recombination. Similarly, any unrelatedDNA that happens to be present when the DNA ends are repaired can beinserted at the target site (FIG. 1G). The different outcomes (FIGS.1E-G) can be obtained simultaneously in the same transformationexperiment.

Any means to make a DNA double-strand break in vivo can be used as theDNA double-strand-break-inducing agent such as the most commonly usedmeganucleases which recognize >18 bp sequences, which are long enough tobe unique in most genomes. Even numerous meganucleases have been foundand characterized to recognize many different sequences, but suchsequences are often not naturally present in important crops such assoybean or maize and even if similar sequences can be found in cropgenomes, the limited numbers of these sequences are still too small tobe useful. Certain meganucleases such as I-CreI can be modified byprotein engineering in such a way that it will no longer preferentiallyrecognize the recognition sequence of wild type I-CreI and instead willpreferentially recognize specifically selected sequences of interest.Taking advantage of the flexibility of the I-CreI endonuclease, one candesign and make a modified I-CreI to cleave a target site of our choicein the genome and subsequently introduce mutations or insert genes ofinterest at the selected target site. The precise genetic engineeringthat this methodology provides will solve many problems that traditionalplant transformation methods such as Agrobacterium infection andbiolistic bombardment currently face, such as unpredictable integration,unwanted endogenous gene interruption, unpredicted transgene expression,etc.

In one embodiment of the invention, we used engineered I-CreI-likemeganucleases that recognize selected different endogenous target sitesin the soybean genome and produced mutations and insertions at theselected target sites.

Example 2 Production of a Complex Trait Locus in the Soybean Genome Neara Transgenic Event for Oil Quality Using Engineered Meganucleases

Soybean lines comprising an endogenous target recognition sequence intheir genome were contacted with a custom designed meganuclease, derivedfrom I CreI, which is designed to specifically recognize and create adouble-strand break in the endogenous target sequence. Soybean embryoscomprising an endogenous target site were contacted with the componentsdescribed below, events selected and characterized.

A. TS21, TS14, TS30 and TS5 Target Sites

Sequence analyses were done for about 500000 bp genomic region insoybean near a transgenic event of interest (event DP-305423-1, U.S.Patent Application Publication No. 2008/0312082 A1, published Dec. 18,2008). A series of soybean genomic endogenous target recognitionsequences, referred to as TS21, TS14, TS30 and TS5, were selected fordesign of custom double-strand break inducing agents derived from I-CreImeganuclease. Each of these target recognition sequences is a unique 22bp polynucleotide. The target recognition sites have the followingsequences:

TS21 target (SEQ ID NO: 1) GGCACTCTCGTGT ^(▾)GTGATTAAA TS14 target(SEQ ID NO: 2) CAGACGTACGCAA ^(▾)GTAGCTTTG TS30 target (SEQ ID NO: 3)GAGTCCCACGCAA ^(▾)GAGCATAAA TS5 target (SEQ ID NO: 4) AAGACTTACGTGT^(▾)GTACTCGTG

The double-strand break sites and overhang regions are shown in bold,the enzyme cuts after C13, as indicated by the solid triangle.

Within the soybean genome, TS5 is about 600 kbp upstream of, and on thesame chromosome as, the transgenic event of interest. TS30, TS21 andTS14 are on the same chromosome as TS5 and are 120 kbp, 125 kbp and 500kbp downstream of the transgenic event of interest (FIG. 2).

B. TS21, TS14, TS30, and TS5 Meganucleases

The I-Crel meganuclease was modified to produce the TS21, TS14, TS30 andTS5 meganucleases, which are designed to recognize their correspondingtarget sequences, under a contract with Precision Biosciences (Durham,N.C. USA). Wild-type I-CreI meganuclease is a homodimer. In order torecognize their target sequences, different substitutions were made toeach monomer. The coding sequences for each monomer were joined by alinker sequence to produce single-chain fusion polypeptides. Genesencoding the designed meganucleases were optimized for expression inplants. SEQ ID NO: 9 is the plant-optimized nucleotide sequence of theTS21 meganuclease. SEQ ID NO: 10 is the plant-optimized nucleotidesequence of the TS14 meganuclease. SEQ ID NO: 11 is the plant-optimizednucleotide sequence of the TS30 meganuclease. SEQ ID NO: 12 is theplant-optimized nucleotide sequence of the TS5 meganuclease. These genesinclude a nucleus localization signal from the SV40 virus (SEQ ID NO:34) and an intron from the potato ST-LS1 gene. The intron preventsexpression of the genes in bacteria during the cloning process, but isnot necessary for expression in plant cells. In these plant-optimizednucleotide sequences(SEQ ID NOs: 9-16) nucleotides 1-30 encode an SV40nucleus localization amino acid sequence, nucleotides 100-261 andnucleotides 850-1011 encode the 1st half and 2nd half target sitebinding amino acid sequences, respectively, nucleotides 403-591 are thepotato ST-LS1 intron, and nucleotides 685-798 encode the amino acidsequence of the polypeptide that links the two re-engineered I-CreImonomers into a single chain.

Plant optimized nucleotide sequences without the ST-LS1 intron encodingthe engineered meganucleases were constructed as well (see, SEQ ID NO:33 for example).

C. Vector Construction for Plant Expression Vectors of the MeganucleaseGenes and Repair DNAs for Transgene Integration by HomologousRecombination

Vectors comprising expression cassettes for the appropriate meganucleasewere constructed using standard molecular biological techniques. Allcustom designed meganucleases were tested including TS21, TS14, TS30 andTS5. For each of the meganucleases, a plant expression vector comprisinga polynucleotide encoding one of the meganuclease genes was operablylinked to a soybean constitutive promoter.

The following meganuclease plant expression vectors were made:

RTW317 (SEQ ID NO: 35, GM-EF1A pro:TS21:pinII) expression cassettecontains the TS21 meganuclease plant optimized sequence without anintron and driven by soybean EF1A promoter.

RTW322 (SEQ ID NO: 36, GM-EF1A pro:TS21 with ST-LS1 intron2:pinII)expression cassette contains the TS21 meganuclease plant optimizedsequence with an intron and driven by soybean EF1A promoter. Otherexpression cassettes were made in a similar manner as RT317 and RTW322,but contained a different promoter, or meganuclease, such as : RTW319(GM-EF1A pro:TS14:pinII), RTW324 (GM-EF1A pro:TS14 with ST-LS1intron2:pinII), RTW323 (GM-EF1A pro:TS5 with ST-LS1 intron2:pinII),RTW325 (GM-EF1A pro:TS30 with ST-LS1 intron2:pinII), RTW345 (GM-UBQpro:TS21:pinII), RTW334(GM-UBQ pro:TS21 with ST-LS1 intron2:pinII),RTW351 (GM-MTH1 pro:TS21:pinII), RTW339 (GM-MTH1 pro:TS21 with ST-LS1intron2:pinII), wherein GM-ETF1A is the soybean ETF1A promoter, GM-UBQis the soybean ubiquitin promoter, GM-MTH1 is the soybean MTH1 promoter,and pinII is the pinII terminator.

To achieve site-specific DNA insertions, a repair DNA (donor DNA)containing the gene of interest has to be simultaneously present in thecell in addition to the target site and the endonuclease. The gene ofinterest was flanked by two homologous recombination fragments (HR1 andHR2), which were 1 to 3 kb long genomic DNA sequences flanking themeganuclease target sites. The gene of interest can be inserted at thetarget site by DNA homologous recombination, a process that isstimulated by the DNA double-strand break at the target site.

A repair DNA (or donor DNA) fragment, Rep-RTW328A (SEQ ID NO: 37) wasmade for gene integration at TS21 target site in the soybean genome. TheRTW328 repair DNA consists of a 1020 bp TS21 HR1 fragment (SEQ IDNO:17), a hygromycin selection marker cassette and a 1000 bp TS21 HR2fragment (SEQ ID NO:18). The hygromycin selection marker was driven by aSCP1 promoter and a NOS terminator (U.S. Pat. No. 6,072,050; Suzuki etal., Gene (2000) 242(1-2):331-336) . Similar repair DNA vectors weremade for TS14, TS30, and TS5 target sites in soybean genome. TheRep-TS14 repair DNA vector consists of a 1000 bp TS14 HR1 fragment (SEQID NO:19, the same hygromycin selection marker cassette and a 928 bpTS14 HR2 fragment (SEQ ID NO:20). The Rep-TS30 repair DNA vector(consists of a 1000 bp TS0 HR1 fragment (SEQ ID NO:21), the samehygromycin selection marker cassette and a 1009 bp TS30 HR2 fragment(SEQ ID NO:22). The Rep-TS5 repair DNA vector consists of a 1006 bp TS5HR1 fragment (SEQ ID NO:23), the same hygromycin selection markercassette and a 1007 bp TS5 HR2 fragment (SEQ ID NO:24).

A DNA double-strand break agent was simultaneously introduced with therepair DNA to facilitate homologous DNA recombination. It is convenientto transiently express the custom designed meganuclease byco-bombardment of a meganuclease expression vector with itscorresponding repair DNA in soybean transformation. The presence orabsence of an ST-LS1 intron in the DNA nucleotide sequence encoding ameganuclease did not affect the functionality of the meganuclease.Alterations at the target site were observed when expression of themeganuclease with both a DNA sequence that included or excluded theST-LS1 intron in the expression cassette.

D. Genomic Sequence Modifications and Transgene Integration atEndogenous Target Sites with Custom Designed Meganucleases

PCR and qPCR assays were done following established protocols usinggene-specific primers and probes (Li et al., (2007) Plant Mol Biol65:329-41; Li et al., (2009) Plant Physiol 151:1087-95). qPCR assaysspecific to the TS21, TS14, TS30, and TS5 target sequences weredeveloped to identify sequence changes that happen in the region. Theprimers and probe were designed as below and tested.

TS21 qPCR:

-   -   Mega21-190F (SEQ ID NO:38)    -   Mega21-301R (SEQ ID NO:39)    -   Mega21-250T (SEQ ID NO:40)

TS14 qPCR:

-   -   Mega14-13F (SEQ ID NO:41)    -   Mega14-128R (SEQ ID NO:42)    -   Mega14-85T (SEQ ID NO:43)

TS30 qPCR:

Mega30-30F (SEQ ID NO:44)

Mega21-87R (SEQ ID NO:45)

Mega21-52T (SEQ ID NO:46)

TS5 qPCR:

-   -   Mega5-F1 (SEQ ID NO:47)    -   Mega5-R1 (SEQ ID NO:48)    -   Mega5-T1 (SEQ ID NO:49)

All hygromycin resistant soybean transgenic events were first analyzedby qPCR assays of the meganuclease target site. Changes in themeganuclease target sequence caused by DNA cleavage and repair result inthe copy number reduction of the meganuclease target site from twocopies in wild type soybean genome to either one or zero copies in thetransgenic events. These “qPCR hit” events with reduced target site copynumbers were chosen for further genomic PCR and sequencing analyses.From qPCR analyses of the TS21, TS14, TS30 and TS5 target sites, it wasshown that the copy numbers of the target sites in most of the positivetransgenic events were reduced by half, indicating one allele of thetarget sites in soybean genome was disrupted by meganuclease cutting/DNArepair mechanism.

Two groups of genomic PCR amplifications were carried out to furthercharacterize these candidate events from qPCR assay to understand thegenomic sequence modifications and transgene integrations. The firstgroup of genomic PCRs were designed to identify mutations in themeganuclease target sites, by amplifying genomic fragments containingthe TS21 target site using a primer that anneals in HR1 and anotherprimer that anneals in HR2. For example, for TS21, the primer set WOL133and WOL134 (SEQ ID NO:50 and 51) were used to amplify genomic fragmentscontaining the TS21 target site (FIG. 3A). The PCR products were clonedand sequenced to identify mutations at the TS21 target site. In somecases, a meganuclease in vitro cutting assay to cut the PCR product ofan unmodified target site was used to test if the target site had beenmodified. In the in vitro cutting assay, the PCR products amplifiedusing primers directed to the target site were digested with themeganuclease at 37° C. overnight. Samples with meganuclease enzyme weretreated with proteinase K and SDS to denature the protein. The digestionproducts were separated on a 1.5 to 2% agarose gel. Undigested productsindicate that the target site was modified. The undigested PCR productswere then cloned and sequenced to verify the genome sequencemodification. An example of the soybean genome sequence modification onTS21target site is shown in FIG. 3B.

With this approach, soybean genome sequence modifications were detectedat TSS, TS14 and TS30 target sites (FIG. 4 and Table 1).

TABLE 1 qPCR copy number analyses of TS30 target sites, pinII(representing the meganuclease cassette) and Hygro (representing therepair DNA cassette) TS30 qPCR pinII qPCR Hygro qPCR Clone ID Copy#copy# copy# A 7052.2.5 0.56 0.00 1.98 A 7052.10.26 0.55 0.00 1.55 A7052.10.28 0.54 0.00 1.96 A 7034.1.11 0.53 0.00 2.98 A 7034.3.1 0.541.70 3.41 A 7034.3.15 0.52 0.96 4.54 WT control 0.96 2.23 5.19

The copy numbers of the TS30 target sites in positive transgenic eventswere reduced by half, indicating one allele of the target sites insoybean genome was disrupted by meganuclease cutting/DNA repairmechanism. These results demonstrate that introduction of themeganuclease gene into the plant cell leads to modifications in thegenomic region of interest.

Both wild type soybean and transgenic embryos have been used in thesoybean transformation. The target modification rate (qPCR) with TS21 isthe same in wild type soybean and the transgenic event. These resultsdemonstrated that we can directly introduce genome modifications in thetransgenic event or introduce genome modifications to the same locus inwild type soybean.

The second group of genomic PCR amplifications was more focused ontransgene integration with border specific PCR. For example, for TS21(FIG. 3A), the primer set WOL190 (SEQ ID NO:52) and WOL242 (SEQ IDNO:53) were designed and used to amplify the left border DNA fragmentthat results from transgene integration. WOL190 is a sequence specificprimer located in soybean genome 5′ beyond the TS21 HR1 region andWOL242 is a sequence specific primer to the 5′ hygromycin-resistancemarker gene coding sequence in the reverse orientation. An 1860 bp PCRproduct can only be obtained when the RTW328A repair DNA is integratedby homologous recombination facilitated by a double-strand breakintroduced at the genomic target site by TS21 meganuclease. Another setof primers, WOL153 (SEQ ID NO:54) and WOL247 (SEQ ID NO: 55), was alsodesigned and used to amplify the right border DNA fragment that resultsfrom transgene integration. WOL153 is the sense primer from the NOSterminator and the WOL247 is a sequence specific primer located insoybean genome 3′ beyond the TS21 HR2 region. A 1727 bp PCR product canonly be obtained when the RTW328A repair DNA is integrated by homologousrecombination facilitated by a double-strand break introduced at thegenomic target site by TS21 meganuclease. Similar genomic PCR primershave been designed and tested for other custom designed meganuclease.

TS21 qPCR

Target site primers

WOL133 (SEQ ID NO:50)

WOL134 (SEQ ID NO:51)

Left border primers

-   -   WOL190 (SEQ ID NO:52)    -   WOL242 (SEQ ID NO:53)

Right border primers

-   -   WOL153 (SEQ ID NO:54)    -   WOL247 (SEQ ID NO:55)

TS14 qPCR

Target site primers

WOL121 (SEQ ID NO:56)

WOL150 (SEQ ID NO:57)

Left border primers

-   -   WOL192 (SEQ ID NO:58)    -   WOL242 (SEQ ID NO:53

Right border primers

-   -   WOL153 (SEQ ID NO:54)    -   WOL193 (SEQ ID NO:59)

TS30 qPCR

Target site primers

WOL113 (SEQ ID NO:60)

WOL114 (SEQ ID NO:61)

Left border primers

-   -   WOL194 (SEQ ID NO:62)    -   WOL242 (SEQ ID NO:53)

Right border primers

-   -   WOL153 (SEQ ID NO:54)    -   WOL195 (SEQ ID NO:63)

TS5 qPCR

Target site primers

WOL105 (SEQ ID NO:64)

WOL144 (SEQ ID NO:65)

Left border primers

-   -   WOL196 (SEQ ID NO:66)    -   WOL242 (SEQ ID NO:53)

Right border primers

-   -   WOL153 (SEQ ID NO:54)    -   WOL197 (SEQ ID NO:67)

Primer pairs were designed with one primer capable of annealing toeither the 5′ or 3′ sequence flanking a target site and another primercapable of annealing to a sequence within the potential insert (i.e.,the transgene). For the TS14 target site, 18 qPCR positive events wereidentified from total 68 events by qPCR analyses. Out of the 18 qPCRpositive events, three events were confirmed to be perfect TS14meganuclease mediated transgene integration events by homologousrecombination.

These results demonstrate that soybean cells possess natural DNA repairmachinery that can repair DNA double-strand break ends by simple endjoining or by homologous recombination. It is thus expected that similarrates of site-directed mutagenesis and gene insertion via homologousrecombination can be achieved at any target sites in the soybean genomeusing proper double-strand break inducing agents specific to the targetrecognition sequences. Using a simple PCR screening procedure describedherein, it is practical to identify such insertion and mutation events.A perfect transgene integration event can be identified when both leftborder PCR and right border PCR indicate insertion at the target site.Transgene integration at the pre-defined target sites within a genomicregion of interest provides a novel gene stacking technology. FIG. 5 isa schematic example of stacking new trait genes into a single targetsite in close proximity to a transgenic event of interest.

Example 3 Production of a Complex Trait Locus in the Soybean Genome Neara Herbicide Resistance Transgenic Event Using Engineered MeganucleasesA. TS7, TS4, TS22 and TS24 Target Sites

The transgene border analyses of a herbicide resistance transgenic event(Event 3560.4.3.5 described in U.S. Patent Application Publication Nos.2010/0184079, 2009/0036308, and 2008/0051288) showed that the transgenewas inserted in a soybean chromosome about 12 cM away from three diseaseresistance markers based on molecular marker analyses (FIG. 6). Sequenceanalyses were done for about 400000 bp in this genomic region ofinterest and four meganuclease target sites (TS7, TS4, TS22 and TS24)were identified with desirable genetic distances between these targetsites and nearby disease resistance markers, and a herbicide resistancetransgenic event. Each of these target recognition sequences is a unique22 bp polynucleotide. The target recognition sites have the followingsequences:

TS7 target (SEQ ID NO: 5) GACATTGTCGTGA ^(▾)GAAAAGAGA TS4 target(SEQ ID NO: 6) AAATCTGTCTTGC ^(▾)GAAACGGCA TS22 target (SEQ ID NO: 7)TATTCTCTCATAA ^(▾)ATAAACTTT TS24 target (SEQ ID NO: 8) GGAATGGACATAA^(▾)GAGAACTGT

The double-strand break sites and overhang regions are shown in bold,the enzyme cuts after C13, as indicated by the solid triangle.

B. TS7, TS4, TS22 and TS24 Meganucleases

The I-CreI meganuclease was modified to produce the TS7, TS4, TS22 andTS24 meganucleases, which are designed to recognize their correspondingtarget sequences, under a contract with Precision Biosciences (Durham,N.C. USA). Wild-type I-Crel meganuclease is a homodimer. In order torecognize their target sequences, different substitutions were made toeach monomer. The coding sequences for each monomer were joined by alinker sequence to produce single-chain fusion polypeptides All thesetarget sites are about 1 to 10 cM away from the cluster of the threedisease resistance markers.

The plant optimized nucleotide sequence encoding the TS7 meganuclease(SEQ ID NO: 13), TS4 meganuclease (SEQ ID NO:14), TS22 meganuclease (SEQID NO:15) and TS24 meganuclease (SEQ ID NO:16) includes a DNA fragment(from bp 1-30) encoding an SV40 nuclear localization signal (MAPKKKRKVH;SEQ ID NO: 34) as well as a ST-LS1 intron (from bp 403 to bp 591 of SEQID 13-16) in order to eliminate expression in E. coli and Agrobacterium.Nucleotides 685-798 of SEQ ID NOs:13-16 encode the amino acid sequenceof the polypeptide that links the two engineered I-Crel monomers into asingle chain. Nucleotides 100-261 of SEQ ID NOs:13-16 and nucleotides850-1011 of SEQ ID NOs:13-16 encode the first half and the second halftarget site binding amino acid sequences, respectively.

C. Vector Construction for Plant Expression Vectors of the MeganucleaseGenes and Repair DNAs for Transgene Integration by HomologousRecombination

Vectors comprising expression cassettes for the appropriate meganucleasewere constructed using standard molecular biological techniques. Allcustom designed meganucleases were tested including TS7, TS4, TS22 andTS24. For each of the meganucleases, a plant expression vectorcomprising a polynucleotide encoding one of the meganuclease genes wasoperably linked to a soybean constitutive promoter.

To achieve site-specific DNA insertions, a repair DNA (donor DNA)containing the DNA of interest has to be simultaneously present in thecell in addition to the target site and the endonuclease. The DNA ofinterest was flanked by two homologous recombination fragments (HR1 andHR2), which were 1 to 3 kb long genomic DNA sequences flanking themeganuclease target sites. The DNA of interest can be inserted at thetarget site by DNA homologous recombination, a process that isstimulated by the DNA double-strand break at the target site.

The HR1 and HR2 domains for TS7, TS4, TS22 and TS24 are SEQ ID NOs: 25and 26, SEQ ID NOs: 27 and 28, SEQ ID NOs: 29 and 30 and SEQ ID NOs: 31and 32, respectively.

Repair DNA vectors were made as described in Example 2 C.

A DNA double-strand break agent was simultaneously introduced with therepair DNA to facilitate homologous DNA recombination. It is convenientto transiently express the custom designed meganuclease byco-bombardment of a meganuclease expression vector with itscorresponding repair DNA in soybean transformation.

Example 4 Cluster of Meganuclease Target Sites in a Short Region of theSoybean Genome for Stacking of Multiple Trait Genes

As shown in FIG. 7, a series of meganuclease target sites can beidentified with desirable genetic distances between these target sites.Custom designed meganucleases can be used to target a series of traitgenes into this defined genome locus either by sequential transformationor by genetic crosses with individual trait genes. Using this methoddepicted in FIG. 7, multiple traits can be stacked in a genomic regionof interest that comprises, for example, a transgene or native gene ofinterest, and other transgenic traits or native trait loci such asdisease resistance markers.

Example 5 Production of a Complex Trait Locus at a Maize EndogenousLocus by Engineered Meganucleases

A. MHP Target Sites

A genomic region encompassing about 1.8 million nucleotides andrepresenting a genetic region of approximately 4.3 centimorgans (cM) ona maize chromosome was chosen as a target region for generation of acomplex trait locus. The genomic region was scanned for 22-mer sequencesthat could serve as target sites containing recognition sequences fordouble-strand-break inducing meganucleases and be useful for insertionof additional transgenes in order to create a complex trait locus. Aseries of 35 putative target sites (SEQ ID NOs: 68-77) were selected ina 2 cM region (FIG. 8) in close proximity of the transgene insertionsite for design of custom double-strand break inducing agents derivedfrom I-CreI meganuclease. FIG. 8 show the genetic and physical locationof the MHP target sites relative to each other and the transgene ofinterest.

B. MHP Meganucleases

The I-CreI meganuclease was modified to produce endonucleases, whichwere designed to recognize their corresponding target sequences, (SEQ IDNOs: 68-77). The design of custom made meganucleases has been describedin United States Patent Application Publication No. US 2007/0117128 A1.

Genes encoding the designed meganucleases were optimized for expressionin plants. The engineered endonuclease expression cassettes containedthe maize codon-optimized nucleotide sequences for better performance inmaize cells. The endonuclease gene sequences were also supplemented withDNA sequences encoding a SV40 nuclear localization signal (SEQ ID NO:34). The maize ubiquitin promoter and the potato proteinase inhibitor IIgene terminator sequences completed the endonuclease gene designs. TheMHP55 (SEQ ID NO:80) expression cassette was additionally modified byaddition of the ST-LS1 intron to the coding sequence of the firstmonomer in order to eliminate its expression in E. coli andAgrobacterium. SEQ ID NO:82 is the plant-optimized nucleotide sequenceof MHP55-2 containing a nuclear localization signal and without anintron. SEQ ID NO: 78 is the plant-optimized nucleotide sequence of theMHP14 meganuclease. A custom designed meganuclease, referred to asMHP14+ was made as well. SEQ ID NO: 79 is the plant-optimized nucleotidesequence of the MHP14+ meganuclease. SEQ ID NO: 83 is theplant-optimized nucleotide sequence of the MHP77 meganuclease

C. Vector Construction for Plant Expression Vectors of the MeganucleaseGenes and Repair (donor) DNAs for Transgene Integration by HomologousRecombination

Vectors comprising expression cassettes for the appropriate meganucleasewere constructed using standard molecular biological techniques. Foreach of the meganucleases, a plant expression vector comprising apolynucleotide encoding one of the meganuclease genes was operablylinked to a maize constitutive promoter.

To achieve site-specific DNA insertions, a repair DNA (donor DNA)containing the gene of interest has to be simultaneously present in thecell in addition to the target site and the meganuclease. A vector(PHP44285 ,SEQ ID NO:104), or PHP44779, SEQ ID NO:105) containing apolynucleotide encoding the engineered meganuclease MHP14, or theoptimized meganuclease MHP14+, and a donor DNA was constructed usingstandard molecular biology techniques. The donor DNA contained anherbicide resistance gene used as the selection marker fortransformation. The herbicide resistance gene MoPAT encodes aphosphinothricin acetyltransferase, and was flanked by two homologousrecombination fragments, HR1 (SEQ ID NO: 84) and HR2 (SEQ ID NO: 85),which were about 1 kb long genomic DNA sequences flanking themeganuclease target sites. Each vector PHP44285 or PHP44779 containedthe meganuclease cassette, the donor DNA and the homology sequences HR1and HR2.

Maize immature embryos 9-12 DAP (days after pollination, approximately1.5-2.0 mm in size) from a maize transformable line were used for genetransformation by bombardment (Example 6). The immature embryos wereplaced on 560Y medium for 4 hours at 26° C. or alternatively, immatureembryos were incubated at temperatures ranging from 26° C. to 37° C. for8 to 24 hours prior to placing on 560Y preceding bombardment (asdescribed in Example 6). Developmental genes ODP2 (AP2 domaintranscription factor ODP2 (Ovule development protein 2); US20090328252A1) and Wushel were included in the experiments through co-bombardment(Example 7). Maize immature embryos were transformed with the vectorsPHP44285 or PHP44779.

D. Genomic Sequence Modifications and Transgene Integration atEndogenous Target Sites with Custom Designed Meganuclease

Successful delivery of the MHP14 donor vector (PHP44285 or PHP44779)conferred bialaphos herbicide resistance, and was used to identifyputative events by callus selection on herbicide containing media.Callus tissues and/or plants regenerated from stable transformants usingstandard culture and regeneration conditions were screened formodification of the endogenous MHP14 target site.

Real time PCR (qPCR) was used to determine the target site copy number.Two copies of the target site indicate that both alleles are wild typeand that no modification occurred at the target site. One copy means oneallele of the target site has changed during repair of the double strandbreak generated by the MHP14 or MHP14+, while absence of the target site(null) is the result of both alleles modified. The copy number can alsobe in between 1 and 2 due to chimeric nature of callus samples. Theprobe sequence for qPCR of MHP14 target site was CAGATTCACGTCAGATTT (SEQID NO: 106), the MHPTS14_forward primer was AGCGACATAGTGGTGTATAAAAGGAA(SEQ ID NO: 107) and MHPTS14_reverse primer wasTGGATTGTAATATGTGTACCTCATGCT (SEQ ID NO: 108). The amplicon wasapproximately 100 bp.

To examine whether increased temperature would increase the rate oftarget site modification, maize embryos were incubated at differenttemperatures following bombardment with several meganucleases. Table 2shows the effect of temperature on the meganuclease activity of MHP14 asdetermined by target site modification. Table 2 indicates that increasedtemperature results in increased target site mutation rate.

TABLE 2 Effect of incubating maize embryos at increased temperaturepost- bombardment on target site mutation rate of meganucleasesMeganuclease Temperature (° C.) Target Site Mutation Rate MHP14 28 14%MHP14 32 46%

Following bombardment, embryos were incubated on 560P (maintenancemedium) for 12 to 48 hours at 28° C. or 32° C. and then placed at 28° C.Herbicide-resistant events were screened for modification at the targetsite by measuring target site copy-number using qPCR. Target sitemutation rate indirectly measures the meganuclease activity. TSMutRate(target site mutation rate) indicated the modification rate of the MHP14or LIG3/4 target site (# events with modification/# events*100%). Asshown in Table 2, target site mutation rate for both MHP14 and LIG34 wasapproximately 3× higher when embryos were placed at 32° C. for 48 hoursafter bombardment compared to no temperature elevation treatment.

Maize calli were also screened for integration of the transgene cassettefrom the donor DNA (PHP44285 or PHP44779) at the MHP14 target sitethrough junction PCR and selected callus events were regenerated into T0plants. FIG. 9A shows an outline of PCR screening for integration of thedonor DNA fragment via homologous recombination at MHP14 target site(PHP44779 donor). Arrows indicate primer locations. FIG. 9B shows PCR ofMHP14 callus events: B1-B12 Junction PCR with primers 146773/146775;b1-b12 Junction PCR with primers 146772/146778. Two events (B2 and B5)yielded the predicted 1-1.2 kb PCR fragments that result fromintegration by homologous recombination for both junctions. PCR productsfrom T0 plants derived from these callus events were sequenced to verifythe callus results. PCR screening revealed integration of the herbicideresistance transgene cassette at MHP14 target site. Primers were fromthe genomic region outside of the homology of donor vector and from thetransgene cassette close to the end of the homology.

FIG. 10A shows a schematic outline of long fragment PCR reactions usedto confirm UBI:moPAT:PinII cassette integration at the endogenous MHP14target . FIG. 10B: shows the results of long fragment PCR on T0 plantsfrom three events where integration occurred at the target site. Theplant A5 was from event #1, A6-A8 event #2, and C4-C6 event #3. 10B-leftshows the long junction fragment PCR on the HR1 side using genomicprimer (146775) and moPAT primer (mopatR2); 10B-right shows the longjunction fragment PCR on HR2 side (mopatF2/146772). Arrows indicated PCRprimer locations. Primer set 146772/mopatF2 amplified a 4 kb fragment,spanning from moPAT gene through the UBI intron, UBI promoter, and theHR2 sequence to the adjacent genomic region. Primer set 146775/mopatR2amplified a 2.2 kb fragment, spanning from the moPAT gene through theHR1 to the adjacent genomic region. These two fragments overlapped andcovered the whole insert at MHP14target site. The sizes of the two longPCR products indicate a perfect integration of the donor gene cassetteat MHP14 target site

To determine the segregation pattern of the integration events inprogeny, T1 seeds from selfed T0 plants were planted in flats and T1plants genotyped by using PCR and/or qPCR. The segregation ratio ofintegration genotypes fit 1:2:1 for wild type (no integration),heterozygous (one allele having integration and the other wild-type) andhomozygous integration of the transgene at the MHP14 target site,demonstrating Mendelian inheritance. No visible phenotype was observedin the homozygous or heterozygous integration plants.

The entire inserted fragment of UBI:moPAT:PinII was obtained by usingPCR on DNA from homozygous T1 plants with primers in the genomic regionoutside of the HR1 and HR2 (146772/146775). A PCR product of 5 kb wasamplified from homozygous plants as expected. A 2 kb PCR product wasamplified from the unmodified intact genomic sequence from wild-typeplants.

Trait gene cassettes can be introduced at other target sites of thecomplex trait locus through homologous recombination mediated byengineered meganucleases. Engineered meganucleases were designed todirect double strand breaks a two other MHP target sites, MHP55 (SEQ IDNO: 72) and MHP77 (SEQ ID NO: 74) within the complex trait locus. Targetsite modification was determined using qPCR. The probe sequence for qPCRscreening of the MHP55 target site was AACCGTCGTGAGACCT (SEQ ID NO:115), the MHPTS55_Forward_MGBprimer sequence was AAGGCGCAGCCGTTGAG (SEQID NO: 116), and MHP55_reverse _MGB primer was CTACCGGTTTCGCGTGCTCT (SEQID NO: 117). The probe sequence for qPCR of MHP77 target site wasTAGTATGACATACATACCGCC (SEQ ID NO: 118), the MHPTS77_Forward_MGB primersequence was TCCTTAGGGCGGTATGTATGTCA (SEQ ID NO: 119), and MHP77_reverse_MGB primer was CATCGGTCAAAAAACACATAAACTTT (SEQ ID NO: 120). The traitgene cassettes encoding MHP14, MHP55 and MHP77 were introduced intomaize somatic embryos via transformation techniques using bombardmentand following bombardment, embryos were incubated on 560P (maintenancemedium) for 48 hours at. As shown in Table 3, maize callus containingthe MHP55 target site bombarded with PHP45782 or PHP46924 which includegenes encoding MHP55 or MHP55.2 meganucleases, respectively, also leadto an observed increase in the target site mutation rate modifiedMHP55.2 variant. In addition, maize callus containing a MHP77 targetsite bombarded with vectors PHP45970 or PHP50238 which include genesencoding MHP77 or MHP77.3 meganucleases, respectively, showed a higherfrequency of mutated target sites from callus bombarded with themodified variant MHP77.3. Taken together, like MHP14, thesemeganucleases directed mutations to their corresponding target sites andmodified versions lead to an increase in the target site mutation rate(approx 2 to 10-fold increase when compared to their original versions)suggesting the newly designed versions of the meganucleases were moreactive than the original nucleases.

TABLE 3 Meganuclease activity (defined as target site mutation rate) oforiginal and modified meganucleases Meganuclease Target Site MutationRate MHP55  0% MHP55-2  5% MHP77  1% MHP77-3 11% MHP14 29% MHP14+ 40%

The mutations observed at these target sites indicated that theengineered meganucleases were functional and that the target sites canbe used for integration of additional trait genes.

E. Production of a Complex Trait Locus at a Maize Endogenous Locus byCrossing

A maize event obtained through random integration containing a transgeneDNA of interest was identified and MHP14, MHP55 and MHP77 target sitessurrounding the transgenic DNA of interest were identified as describedabove. Other maize events containing a modification at the MHP14, MHP55and MHP77 target site (through addition of herbicide resistance gene asdescribed above) were also identified.

Plants homozygous for the integration of a herbicide resistance gene atthe MHP14 target site were crossed with homozygous maize plantscontaining the transgene DNA of interest. The cross resulted in fertileplants producing F1 seeds. The F1 seeds were planted and out-crossedwith Elite inbred line plants and screened for the stacked phenotype.Additional trait genes can be added to the complex trait locus bycrossing one transgenic event containing n-transgenes with othertrangenic events containing the additional trait gene at the additionaltarget site, and progeny can be screened for the presence of n+1transgenes. This process can be repeated as many times as the amount oftarget sites are present in the complex trait locus.

F. Production of a Complex Trait Locus at a Maize Endogenous Locus bySerial Transformation

A complex trait locus can be also be created by serial transformation. Afirst transformed line containing a first trait gene integrated at afirst MHP target site can be used to supply embryos. The firsttransformed line can be retransformed with a second trait gene and avector encoding a second engineered meganuclease; resulting in thesecond trait gene being integrated at a second MHP target site throughhomologous recombination mediated by the second engineered meganuclease.The homozygous integration plants containing a selectable marker at theMHP14 target site can be used to supply embryos. Two rounds oftransformations will create two traits at the MHP locus. A transformedline that is homozygous for integration events with two trait genes atMHP target sites can be used to supply embryos for anotherretransformation, and a third trait gene can be introduced to a thirdtarget site.

Example 6 Transformation of Maize Immature Embryos

Transformation can be accomplished by various methods known to beeffective in plants, including particle-mediated delivery,Agrobacterium-mediated transformation, PEG-mediated delivery, andelectroporation.

a. Particle-Mediated Delivery

Transformation of maize immature embryos using particle delivery isperformed as follows. Media recipes follow below.

The ears are husked and surface sterilized in 30% Clorox bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are isolated and placed embryo axis sidedown (scutellum side up), 25 embryos per plate, on 560Y medium for 4hours and then aligned within the 2.5-cm target zone in preparation forbombardment. Alternatively, isolated embryos are placed on 560L(Initiation medium) and placed in the dark at temperatures ranging from26° C. to 37° C. for 8 to 24 hours prior to placing on 560Y for 4 hoursat 26° C. prior to bombardment as described above.

A plasmid comprising the Zm-BBM (also referred to as Zm-ODP2) codingsequence (set forth in SEQ ID NO: 9) operably linked to a promoter isconstructed. This could be a weak promoter such as nos, atissue-specific promoter, such as globulin-1 or oleosin, an induciblepromoter such as In2, or a strong promoter such as ubiquitin plus aplasmid containing the selectable marker gene phosphinothricinN-acetyltransferase (PAT; Wohlleben et al. (1988) Gene 70:25 37) thatconfers resistance to the herbicide bialaphos. Furthermore, plasm idscontaining the double strand brake inducing agent and donor DNA such asPHP44285 or PHP44779 are constructed as described above andco-bombareded with the plasmids containing the developmental genes ODP2(AP2 domain transcription factor ODP2 (Ovule development protein 2);US20090328252 A1) and Wushel.

The plasmids are precipitated onto 1.1 μm (average diameter) tungstenpellets using a calcium chloride (CaCl₂) precipitation procedure bymixing 100 μl prepared tungsten particles in water, 10 μl (1 μg) DNA inTris EDTA buffer (1 μg total DNA), 100 μl 2.5 M CaC12, and 10 μl 0.1 Mspermidine. Each reagent is added sequentially to the tungsten particlesuspension, with mixing. The final mixture is sonicated briefly andallowed to incubate under constant vortexing for 10 minutes. After theprecipitation period, the tubes are centrifuged briefly, liquid isremoved, and the particles are washed with 500 ml 100% ethanol, followedby a 30 second centrifugation. Again, the liquid is removed, and 105 μl100% ethanol is added to the final tungsten particle pellet. Forparticle gun bombardment, the tungsten/DNA particles are brieflysonicated. 10 μl of the tungsten/DNA particles is spotted onto thecenter of each macrocarrier, after which the spotted particles areallowed to dry about 2 minutes before bombardment.

The sample plates are bombarded at level #4 with a Biorad Helium Gun.All samples receive a single shot at 450 PSI, with a total of tenaliquots taken from each tube of prepared particles/DNA.

Following bombardment, the embryos are incubated on 560P (maintenancemedium) for 12 to 48 hours at temperatures ranging from 26C to 37C, andthen placed at 26C. After 5 to 7 days the embryos are transferred to560R selection medium containing 3 mg/liter Bialaphos, and subculturedevery 2 weeks at 26C. After approximately 10 weeks of selection,selection-resistant callus clones are transferred to 288J medium toinitiate plant regeneration. Following somatic embryo maturation (2-4weeks), well-developed somatic embryos are transferred to medium forgermination and transferred to a lighted culture room. Approximately7-10 days later, developing plantlets are transferred to 272Vhormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto a 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to Classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for transformation efficiency, and/ormodification of regenerative capabilities.

Initiation medium (560L) comprises 4.0 g/I N6 basal salts (SIGMAC-1416), 1.0 ml/I Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/Ithiamine HCl, 20.0 g/I sucrose, 1.0 mg/I 2,4-D, and 2.88 g/I L-proline(brought to volume with D-I H2O following adjustment to pH 5.8 withKOH); 2.0 g/I Gelrite (added after bringing to volume with D-I H2O); and8.5 mg/I silver nitrate (added after sterilizing the medium and coolingto room temperature).

Maintenance medium (560P) comprises 4.0 g/I N6 basal salts (SIGMAC-1416), 1.0 ml/I Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/Ithiamine HCl, 30.0 g/I sucrose, 2.0 mg/I 2,4-D, and 0.69 g/I L-proline(brought to volume with D-I H2O following adjustment to pH 5.8 withKOH); 3.0 g/I Gelrite (added after bringing to volume with D-I H2O); and0.85 mg/I silver nitrate (added after sterilizing the medium and coolingto room temperature).

Bombardment medium (560Y) comprises 4.0 g/I N6 basal salts (SIGMAC-1416), 1.0 ml/I Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/Ithiamine HCl, 120.0 g/I sucrose, 1.0 mg/I 2,4-D, and 2.88 g/I L-proline(brought to volume with D-I H2O following adjustment to pH 5.8 withKOH); 2.0 g/I Gelrite (added after bringing to volume with D-I H2O); and8.5 mg/I silver nitrate (added after sterilizing the medium and coolingto room temperature).

Selection medium (560R) comprises 4.0 g/I N6 basal salts (SIGMA C-1416),1.0 ml/I Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/I thiamineHCl, 30.0 g/I sucrose, and 2.0 mg/I 2,4-D (brought to volume with D-IH2O following adjustment to pH 5.8 with KOH); 3.0 g/I Gelrite (addedafter bringing to volume with D-I H2O); and 0.85 mg/I silver nitrate and3.0 mg/I bialaphos (both added after sterilizing the medium and coolingto room temperature).

Plant regeneration medium (288J) comprises 4.3 g/I MS salts (GIBCO11117-074), 5.0 ml/I MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/I thiamine HCL, 0.10 g/I pyridoxine HCL, and 0.40 g/I glycinebrought to volume with polished D-I H2O) (Murashige and Skoog (1962)Physiol. Plant. 15:473), 100 mg/I myo-inositol, 0.5 mg/I zeatin, 60 g/Isucrose, and 1.0 ml/I of 0.1 mM abscisic acid (brought to volume withpolished D-I H2O after adjusting to pH 5.6); 3.0 g/I Gelrite (addedafter bringing to volume with D-I H2O); and 1.0 mg/I indoleacetic acidand 3.0 mg/I bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/I MS salts (GIBCO11117-074), 5.0 ml/I MS vitamins stock solution (0.100 g/I nicotinicacid, 0.02 g/I thiamine HCL, 0.10 g/I pyridoxine HCL, and 0.40 g/Iglycine brought to volume with polished D-I H2O), 0.1 g/I myo-inositol,and 40.0 g/I sucrose (brought to volume with polished D-I H2O afteradjusting pH to 5.6); and 6 g/I bacto-agar (added after bringing tovolume with polished D-I H2O), sterilized and cooled to 60° C.

b. Agrobacterium-mediated transformation

Agrobacterium-mediated transformation was performed essentially asdescribed in Djukanovic et al. (2006) Plant Biotech J 4:345-57. Briefly,10-12 day old immature embryos (0.8 -2.5 mm in size) were dissected fromsterilized kernels and placed into liquid medium (4.0 g/L N6 Basal Salts(Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix (Sigma E-1511), 1.0 mg/Lthiamine HCl, 1.5 mg/L 2, 4-D, 0.690 g/L L-proline, 68.5 g/L sucrose,36.0 g/L glucose, pH 5.2). After embryo collection, the medium wasreplaced with 1 ml Agrobacterium at a concentration of 0.35-0.45 OD550.Maize embryos were incubated with Agrobacterium for 5 min at roomtemperature, then the mixture was poured onto a media plate containing4.0 g/L N6 Basal Salts (Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix(Sigma E-1511), 1.0 mg/L thiamine HCl, 1.5 mg/L 2, 4-D, 0.690 g/LL-proline, 30.0 g/L sucrose, 0.85 mg/L silver nitrate, 0.1 nMacetosyringone, and 3.0 g/L Gelrite, pH 5.8. Embryos were incubated axisdown, in the dark for 3 days at 20° C., then incubated 4 days in thedark at 28° C., then transferred onto new media plates containing 4.0g/L N6 Basal Salts (Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix(Sigma E-1511), 1.0 mg/L thiamine HCl, 1.5 mg/L 2, 4-D, 0.69 g/LL-proline, 30.0 g/L sucrose, 0.5 g/L MES buffer, 0.85 mg/L silvernitrate, 3.0 mg/L Bialaphos, 100 mg/L carbenicillin, and 6.0 g/L agar,pH 5.8. Embryos were subcultured every three weeks until transgenicevents were identified. Somatic embryogenesis was induced bytransferring a small amount of tissue onto regeneration medium (4.3 g/LMS salts (Gibco 11117), 5.0 ml/L MS Vitamins Stock Solution, 100 mg/Lmyo-inositol, 0.1 μM ABA, 1 mg/L IAA, 0.5 mg/L zeatin, 60.0 g/L sucrose,1.5 mg/L Bialaphos, 100 mg/L carbenicillin, 3.0 g/L Gelrite, pH 5.6) andincubation in the dark for two weeks at 28° C. All material with visibleshoots and roots were transferred onto media containing 4.3 g/L MS salts(Gibco 11117), 5.0 ml/L MS Vitamins Stock Solution, 100 mg/Lmyo-inositol, 40.0 g/L sucrose, 1.5 g/L Gelrite, pH 5.6, and incubatedunder artificial light at 28° C. One week later, plantlets were movedinto glass tubes containing the same medium and grown until they weresampled and/or transplanted into soil.

Example 7 Transient Expression of BBM Enhances Transformation

Parameters of the transformation protocol can be modified to ensure thatthe BBM activity is transient. One such method involves precipitatingthe BBM-containing plasmid in a manner that allows for transcription andexpression, but precludes subsequent release of the DNA, for example, byusing the chemical PEI. In one example, the BBM plasmid is precipitatedonto gold particles with PEI, while the transgenic expression cassette(UBI:moPAT˜GFPm:PinII; moPAT is the maize optimized PAT gene) to beintegrated is precipitated onto gold particles using the standardcalcium chloride method.

Briefly, gold particles were coated with PEI as follows. First, the goldparticles were washed. Thirty-five mg of gold particles, 1.0 in averagediameter (A.S.I. #162-0010), were weighed out in a microcentrifuge tube,and 1.2 ml absolute EtOH was added and vortexed for one minute. The tubewas incubated for 15 minutes at room temperature and then centrifuged athigh speed using a microfuge for 15 minutes at 4oC. The supernatant wasdiscarded and a fresh 1.2 ml aliquot of ethanol (EtOH) was added,vortexed for one minute, centrifuged for one minute, and the supernatantagain discarded (this is repeated twice). A fresh 1.2 ml aliquot of EtOHwas added, and this suspension (gold particles in EtOH) was stored at−20oC for weeks. To coat particles with polyethylimine (PEI; Sigma #P3143), 250 μl of the washed gold particle/EtOH mix was centrifuged andthe EtOH discarded. The particles were washed once in 100 μl ddH2O toremove residual ethanol, 250 μl of 0.25 mM PEI was added, followed by apulse-sonication to suspend the particles and then the tube was plungedinto a dry ice/EtOH bath to flash-freeze the suspension, which was thenlyophilized overnight. At this point, dry, coated particles could bestored at −80oC for at least 3 weeks. Before use, the particles wererinsed 3 times with 250 μl aliquots of 2.5 mM HEPES buffer, pH 7.1, with1× pulse-sonication, and then a quick vortex before each centrifugation.The particles were then suspended in a final volume of 250 μl HEPESbuffer. A 25 μl aliquot of the particles was added to fresh tubes beforeattaching DNA. To attach uncoated DNA, the particles werepulse-sonicated, then 1 μg of DNA (in 5 μl water) was added, followed bymixing by pipetting up and down a few times with a Pipetteman andincubated for 10 minutes. The particles were spun briefly (i.e. 10seconds), the supernatant removed, and 60 μl EtOH added. The particleswith PEI-precipitated DNA-1 were washed twice in 60 μl of EtOH. Theparticles were centrifuged, the supernatant discarded, and the particleswere resuspended in 45 μl water. To attach the second DNA (DNA-2),precipitation using TFX-50 was used. The 45 μl of particles/DNA-1suspension was briefly sonicated, and then 5 μl of 100 ng/μl of DNA-2and 2.5 μl of TFX-50 were added. The solution was placed on a rotaryshaker for 10 minutes, centrifuged at 10,000 g for 1 minute. Thesupernatant was removed, and the particles resuspended in 60 μl of EtOH.The solution was spotted onto macrocarriers and the gold particles ontowhich DNA-1 and DNA-2 had been sequentially attached were delivered intoscutellar cells of 10 DAP Hi-II immature embryos using a standardprotocol for the PDS-1000. For this experiment, the DNA-1 plasmidcontained a UBI:RFP:pinII expression cassette, and DNA-2 contained aUBI:CFP:pinII expression cassette. Two days after bombardment, transientexpression of both the CFP and RFP fluorescent markers was observed asnumerous red & blue cells on the surface of the immature embryo. Theembryos were then placed on non-selective culture medium and allowed togrow for 3 weeks before scoring for stable colonies. After this 3-weekperiod, 10 multicellular, stably-expressing blue colonies were observed,in comparison to only one red colony. This demonstrated thatPEI-precipitation could be used to effectively introduce DNA fortransient expression while dramatically reducing integration of thePEI-introduced DNA and thus reducing the recovery of RFP-expressingtransgenic events. In this manner, PEI-precipitation can be used todeliver transient expression of BBM and/or WUS2.

For example, the particles are first coated with UBI:BBM:pinII usingPEI, then coated with UBI:moPAT˜YFP using TFX-50, and then bombardedinto scutellar cells on the surface of immature embryos. PEI-mediatedprecipitation results in a high frequency of transiently expressingcells on the surface of the immature embryo and extremely lowfrequencies of recovery of stable transformants (relative to the TFX-50method). Thus, it is expected that the PEI-precipitated BBM cassetteexpresses transiently and stimulates a burst of embryogenic growth onthe bombarded surface of the tissue (i.e. the scutellar surface), butthis plasmid will not integrate. The PAT˜GFP plasmid released from theCa++/gold particles is expected to integrate and express the selectablemarker at a frequency that results in substantially improved recovery oftransgenic events. As a control treatment, PEI-precipitated particlescontaining a UBI:GUS:pinII (instead of BBM) are mixed with thePAT-GFP/Ca++ particles. Immature embryos from both treatments are movedonto culture medium containing 3 mg/I bialaphos. After 6-8 weeks, it isexpected that GFP+, bialaphos-resistant calli will be observed in thePEI/BBM treatment at a much higher frequency relative to the controltreatment (PEI/GUS).

As an alternative method, the BBM plasmid is precipitated onto goldparticles with PEI, and then introduced into scutellar cells on thesurface of immature embryos, and subsequent transient expression of theBBM gene elicits a rapid proliferation of embryogenic growth. Duringthis period of induced growth, the explants are treated withAgrobacterium using standard methods for maize (see Example 1), withT-DNA delivery into the cell introducing a transgenic expressioncassette such as UBI:moPAT˜GFPm:pinII. After co-cultivation, explantsare allowed to recover on normal culture medium, and then are moved ontoculture medium containing 3 mg/I bialaphos. After 6-8 weeks, it isexpected that GFP+, bialaphos-resistant calli will be observed in thePEI/BBM treatment at a much higher frequency relative to the controltreatment (PEI/GUS).

It may be desirable to “kick start” callus growth by transientlyexpressing the BBM and/or WUS2 polynucleotide products. This can be doneby delivering BBM and WUS2 5′-capped polyadenylated RNA, expressioncassettes containing BBM and WUS2 DNA, or BBM and/or WUS2 proteins. Allof these molecules can be delivered using a biolistics particle gun. Forexample 5′-capped polyadenylated BBM and/or WUS2 RNA can easily be madein vitro using Ambion's mMessage mMachine kit. RNA is co-delivered alongwith DNA containing a polynucleotide of interest and a marker used forselection/screening such as Ubi:moPAT˜GFPm:PinII. It is expected thatthe cells receiving the RNA will immediately begin dividing more rapidlyand a large portion of these will have integrated the agronomic gene.These events can further be validated as being transgenic clonalcolonies because they will also express the PAT˜GFP fusion protein (andthus will display green fluorescence under appropriate illumination).Plants regenerated from these embryos can then be screened for thepresence of the polynucleotide of interest.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1.-91. (canceled)
 92. A maize plant comprising a complex transgenictrait locus in a plant, the trait locus comprising at least first andsecond altered target sequences, wherein the first altered targetsequence originated from a first endogenous target sequence that isrecognized and cleaved by a first double-strand-break-inducing agent andthe second altered target sequence originated from a second endogenoustarget sequence that is recognized and cleaved by a seconddouble-strand-break-inducing agent, wherein each of said altered targetsequences differ from their corresponding endogenous target sequence,wherein the first and second endogenous target sequences are located onthe same arm of the same chromosome, wherein each of the alterationscomprises a heterologous polynucleotide, and wherein at least one of thedouble-strand-break-inducing agents cleaves SEQ ID NO:
 75. 93. The maizeplant of claim 1 wherein the heterologous polynucleotide is selectedfrom the group consisting of: DNA for gene silencing, DNA encoding aphenotypic marker, and DNA encoding a protein providing an agronomicadvantage.
 94. A seed of the maize plant of claim 1, comprising saidcomplex transgenic trait locus of claim 1.